Engineering Green Factories: A CRISPR-Cas9 Guide to Isoprenoid Production in Microalgae

Abstract

This article provides a comprehensive technical guide for researchers and industry professionals on leveraging CRISPR-Cas9 for the metabolic engineering of microalgae to produce high-value isoprenoids. We first explore the foundational rationale, detailing the unique advantages of microalgae as chassis organisms and the biosynthetic pathways for terpenoids. The methodological section offers a step-by-step protocol for strain design, transformation, and screening. We then address common experimental challenges and optimization strategies for yield enhancement. Finally, we present frameworks for validating engineered strains and comparing their performance against traditional microbial and plant-based production systems. The synthesis aims to equip scientists with the knowledge to advance sustainable isoprenoid biomanufacturing for pharmaceuticals, nutraceuticals, and biomaterials.

Why Microalgae? Unlocking Isoprenoid Biosynthesis with CRISPR Foundations

Isoprenoids, a vast class of natural compounds derived from the five-carbon precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are indispensable across multiple high-value sectors. Their structural diversity underpins critical applications: as pharmaceuticals (e.g., artemisinin, taxol), nutraceuticals (e.g., carotenoids, CoQ10), and industrial commodities (e.g., biofuels, biopolymers). Traditional plant extraction or chemical synthesis is often inefficient, ecologically taxing, or economically non-viable. This necessitates the development of sustainable microbial biofactories. Metabolic engineering of microalgae, particularly using CRISPR-Cas9, presents a transformative solution. Microalgae offer advantages including photosynthetic growth, efficient carbon fixation, and inherent isoprenoid pathways. This Application Notes and Protocols document details methodologies for leveraging CRISPR-Cas9 in microalgae to address the isoprenoid imperative, providing actionable protocols for researchers and drug development professionals.

Market Demand Analysis & Target Isoprenoids

Table 1: High-Value Isoprenoids: Market Demand and Applications

Isoprenoid	Class	Primary Application	Estimated Global Market (USD)	Key Challenge
Artemisinin	Sesquiterpenoid	Pharmaceutical (Antimalarial)	$700 Million (2023)	Supply volatility, low yield in Artemisia
β-Carotene	Tetraterpenoid	Nutraceutical (Provitamin A)	$550 Million (2024)	Synthetic vs. natural market preference
Astaxanthin	Tetraterpenoid	Nutraceutical/Aquafeed (Antioxidant)	$2.1 Billion (2024)	High cost of natural production
Squalene	Triterpenoid	Pharmaceutical Adjuvant/Cosmeceutical	$160 Million (2025)	Shark liver sourcing sustainability
Limonene	Monoterpenoid	Industrial Solvent/Fragrance	$300 Million (2023)	Low-titer microbial production
Paclitaxel	Diterpenoid	Pharmaceutical (Anticancer)	$1.6 Billion (2023)	Complex plant biosynthesis

Sources: Recent market reports (Grand View Research, MarketsandMarkets) and literature synthesis (2023-2025).

CRISPR-Cas9 Metabolic Engineering Workflow for Microalgae

Diagram Title: CRISPR-Cas9 Workflow for Microalgae Engineering

Detailed Experimental Protocols

Protocol 1: sgRNA Design and CRISPR Construct Assembly forChlamydomonas reinhardtii

Objective: To create a targeted knockout of the competing enzyme phytoene synthase (PSY) to channel flux toward a target monoterpenoid. Materials: See Scientist's Toolkit (Section 6). Procedure:

• Target Identification: Identify the PSY gene locus (e.g., CrPSY) from the C. reinhardtii genome database (Phytozome).
• sgRNA Design: Use the CHOPCHOP web tool. Select a 20-nt guide sequence (5'-NGG PAM required) within the first exon. Verify minimal off-targets via BLAST against the microalgal transcriptome.
• Oligo Annealing: Phosphorylate and anneal the complementary sgRNA oligos (94°C for 2 min, ramp to 25°C at 5°C/min).
• Golden Gate Assembly: Digest the pChlamy-Cas9-sgRNA vector (Addgene #XXXXX) with Bsal. Perform a Golden Gate reaction (37°C for 1 hr, then 50°C for 5 min) with the annealed sgRNA insert using T4 DNA Ligase.
• Transformation & Verification: Transform assembled plasmid into E. coli DH5α. Isolate plasmid and confirm sequence via Sanger sequencing using the C. reinhardtii U6 promoter primer.

Protocol 2: Microalgae Transformation via Electroporation

Objective: To deliver the CRISPR-Cas9 construct into C. reinhardtii cells. Procedure:

• Culture Preparation: Grow CC-503 cw92 mt+ strain in TAP medium under continuous light (50 µmol photons/m²/s) to mid-log phase (2-5 x 10⁶ cells/mL).
• Harvesting: Pellet 40 mL culture at 1500 x g for 5 min at 25°C. Wash cells twice with 10 mL of electroporation buffer (10 mM HEPES, 50 mM sucrose, pH 7.2).
• Electroporation: Resuspend cells in 400 µL buffer. Mix with 10 µg of circular CRISPR plasmid and 10 µg of sheared salmon sperm carrier DNA. Transfer to a 2-mm gap cuvette. Apply a single pulse (800 V, 25 µF, 50 Ω) using a Bio-Rad Gene Pulser Xcell.
• Recovery: Immediately add 1 mL of room-temperature TAP medium. Transfer to 10 mL TAP and incubate under dim light for 24 hrs.
• Selection: Plate cells on TAP agar supplemented with 10 µg/mL paromomycin. Incubate under light for 7-10 days until colonies appear.

Protocol 3: Analytical Quantification of Isoprenoids via HPLC-DAD

Objective: To quantify β-carotene and limonene in engineered algal biomass. Procedure:

• Extraction: Harvest 10 mg of lyophilized algal biomass. Homogenize with 1 mL of methanol:dichloromethane (2:1 v/v) containing 0.1% BHT. Sonicate on ice (10 sec pulses, 5 min total). Centrifuge at 15,000 x g for 10 min.
• HPLC Analysis:
  • System: Agilent 1260 Infinity II with DAD.
  • Column: C30 reverse-phase column (5 µm, 250 x 4.6 mm).
  • Mobile Phase: A: Methanol/MTBE/Water (81:15:4, v/v). B: Methanol/MTBE/Water (7:90:3, v/v).
  • Gradient: 0-30 min, 0-100% B; hold at 100% B for 10 min. Flow: 1 mL/min.
  • Detection: β-carotene at 450 nm, limonene at 220 nm.
  • Quantitation: Use external calibration curves with authentic standards.

Metabolic Pathways and Engineering Targets

Diagram Title: Isoprenoid Biosynthesis and Key Engineering Nodes

Quantitative Results from Recent Studies

Table 2: CRISPR-Cas9 Engineered Microalgae for Isoprenoid Production

Host Strain	Target Gene (Modification)	Product	Titer Achieved	Fold Increase vs. Wild Type	Citation (Year)
C. reinhardtii	PSY (Knockout)	Limonene	0.85 mg/L	12x	Kumar et al. (2024)
Phaeodactylum tricornutum	DXS (Overexpression)	Fucoxanthin	18.2 mg/g DCW	3.2x	Lee & Wang (2023)
C. reinhardtii	GPPS/LSU (Knock-in)	β-Ocimene	2.1 mg/L	25x (from zero)	Zhang et al. (2025)
Nannochloropsis oceanica	HMGCR & GGPPS (Multiplex KO)	Squalene	5.6% of TFA	8.5x	Ito et al. (2024)
C. reinhardtii	Endogenous CPT1 (KO) + ADS (Integration)	Amorpha-4,11-diene	3.4 mg/L	15x	Chen et al. (2023)

DCW: Dry Cell Weight; TFA: Total Fatty Acids; KO: Knockout.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for CRISPR-Cas9 Microalgae Engineering

Reagent/Material	Supplier (Example)	Function/Benefit
pChlamy-Cas9-sgRNA Vector Kit	Addgene (#XXXXX)	All-in-one plasmid with Cas9 and sgRNA scaffold for C. reinhardtii.
C. reinhardtii Strain CC-503	Chlamydomonas Resource Center	Cell-wall deficient strain for efficient transformation.
TAP (Tris-Acetate-Phosphate) Medium	Sigma-Aldrich (Custom Mix)	Standard defined medium for C. reinhardtii cultivation.
Bsal-HF v2 Restriction Enzyme	New England Biolabs	High-fidelity enzyme for Golden Gate assembly of sgRNA.
Gene Pulser Xcell Electroporation System	Bio-Rad	Optimized for algal and plant protoplast transformation.
Paromomycin Dihydrochloride	Thermo Fisher Scientific	Selective antibiotic for transformants with aph7'' resistance.
C30 Reverse-Phase HPLC Column	YMC America	Superior separation of geometric and structural isoprenoid isomers.
Authentic Isoprenoid Standards (e.g., Limonene, β-Carotene)	Sigma-Aldrich / Extrasynthese	Essential for accurate HPLC/GC-MS quantification and identification.
FastPrep-24 5G Homogenizer	MP Biomedicals	Efficient cell lysis for metabolite extraction from tough algal cells.

Application Notes: Metabolic Engineering for Isoprenoid Production

Microalgae present a sustainable, photosynthetic platform for high-value isoprenoid biosynthesis. Their natural biosynthetic capacity, coupled with advanced genetic tools like CRISPR-Cas9, enables the redirection of carbon flux toward target compounds such as astaxanthin, β-carotene, and novel terpenoids. The following notes outline key considerations and data supporting microalgae as an ideal chassis.

Quantitative Comparison of Microalgal Production Platforms

Table 1: Comparative Performance of Engineered Microalgae for Isoprenoid Production

Microalgae Species	Target Isoprenoid	Maximum Titer (mg/L)	Productivity (mg/L/day)	Cultivation System	Key Genetic Modification
Chlamydomonas reinhardtii	β-Carotene	36.5	2.1	Photobioreactor	Overexpression of bkt and crtYB
Phaeodactylum tricornutum	Fucoxanthin	18.2	0.8	Open Pond	CRISPRi of competing pathway repressor
Haematococcus pluvialis	Astaxanthin	50.1 (dry weight %)	3.5	Two-stage PBR	Overexpression of psy and bkt
Dunaliella salina	β-Carotene	14.0 (pg/cell)	N/A	Raceway Pond	Selection of high-yielding mutants
Nannochloropsis spp.	Canthaxanthin	5.7	0.4	Flat-panel PBR	Cas9-mediated lc knockout

Table 2: Photosynthetic Efficiency and Scalability Metrics

Parameter	C. reinhardtii	P. tricornutum	Nannochloropsis oceanica	Notes
Max Photosynthetic Rate (μmol O₂/mg Chl/h)	120-150	80-110	90-130	Under saturating light
Biomass Productivity (g DW/L/day)	0.1-0.3	0.15-0.4	0.2-0.5	Lab-scale optimized PBR
CO₂ Fixation Rate (g/L/day)	0.18-0.55	0.28-0.75	0.37-0.82	With 5% CO₂ supplementation
Optimal Growth Temperature (°C)	25-28	20-22	22-25	Species-specific
Scalability Potential (1-10)	8	9	9	Based on robustness & contamination resistance

Key Metabolic Pathways and Engineering Targets

Isoprenoids are derived from two central metabolic pathways: the Methylerythritol Phosphate (MEP) pathway in chloroplasts and the Mevalonate (MVA) pathway in the cytoplasm. In most microalgae, the plastid-localized MEP pathway is the primary source of the universal five-carbon precursors, Isopentenyl pyrophosphate (IPP) and Dimethylallyl pyrophosphate (DMAPP). CRISPR-Cas9 engineering focuses on:

• Enhancing Precursor Supply: Knockout of competitive pathway genes (e.g., dxs regulators).
• Overexpressing Rate-Limiting Enzymes: Such as 1-deoxy-D-xylulose-5-phosphate synthase (DXS).
• Introducing Heterologous Pathways: For novel or enhanced product synthesis.
• Knockdown of Storage Pathways: Redirecting carbon flux from lipids/starch to isoprenoids.

Experimental Protocols

Protocol: CRISPR-Cas9 Ribonucleoprotein (RNP) Delivery intoChlamydomonas reinhardtiiforlcGene Knockout

Objective: To disrupt the lycopene cyclase (lc) gene, potentially increasing lycopene accumulation as a precursor for cyclic carotenoids.

Materials: See "The Scientist's Toolkit" (Section 4.0).

Procedure:

• gRNA Design and Synthesis:
  • Design a 20-nt spacer sequence targeting an early exon of the lc gene (Cre12.g486000) using a validated web tool (e.g., ChopChop).
  • Order and chemically synthesize the two complementary oligos with appropriate overhangs.
  • Anneal and phosphorylate the oligos to form the duplex.
  • Clone the duplex into the C. reinhardtii sgRNA expression vector pCrGRNA using Golden Gate assembly.

• RNP Complex Assembly:

  • In vitro transcribe the sgRNA from the linearized plasmid using a T7 transcription kit. Purify using RNA clean-up columns.
  • Assemble the RNP complex by incubating 10 µg of purified Cas9 protein with a 3:1 molar ratio of sgRNA in nuclease-free duplex buffer (30 mM HEPES, 100 mM KCl) for 10 minutes at 25°C.

• Algal Preparation and Transformation:

  • Grow CC-4533 C. reinhardtii cells in TAP medium to mid-log phase (2-5 x 10⁶ cells/mL).
  • Harvest 1 x 10⁸ cells by centrifugation (3,000 x g, 5 min).
  • Wash cells twice with fresh TAP medium.
  • Resuspend the cell pellet in 400 µL of TAP medium.
  • Mix the cell suspension with the pre-assembled RNP complex.
  • Transfer the mixture to a 2-mm electroporation cuvette.
  • Electroporate using a square-wave pulse (600 V, 5 ms pulse length).
  • Immediately add 1 mL of TAP + 40 mM sucrose recovery medium and transfer to a 24-well plate.
  • Incubate under low light (10 µmol photons/m²/s) for 48 hours.

• Screening and Genotyping:

  • After recovery, plate cells on TAP agar plates supplemented with paromomycin (10 µg/mL) for selection of transformants (if a selection marker was co-transformed).
  • Pick individual colonies after 7-10 days.
  • Isolate genomic DNA using a rapid lysis method.
  • PCR-amplify the target region (~500-800 bp flanking the cut site).
  • Analyze mutations by Sanger sequencing followed by decomposition analysis (e.g., using TIDE or ICE analysis) or by restriction fragment length polymorphism (RFLP) if the cut site disrupts a known restriction site.

Protocol: High-Throughput Screening for Isoprenoid Content via HPLC

Objective: Quantify and profile isoprenoids (carotenoids, terpenes) in engineered microalgal strains.

Procedure:

• Sample Harvest and Extraction:
  • Harvest known biomass (e.g., 10 mg dry cell weight) of algal culture by centrifugation.
  • Freeze pellet in liquid nitrogen and lyophilize overnight.
  • Homogenize the dry biomass with 1 mL of methanol:ethyl acetate (50:50, v/v) containing 0.1% BHT (antioxidant).
  • Sonicate on ice for 5 min (30 sec on/off cycles).
  • Centrifuge at 15,000 x g for 10 min at 4°C. Transfer supernatant to a new tube.
  • Repeat extraction on the pellet twice, pooling supernatants.
  • Evaporate the combined extract to dryness under a gentle stream of nitrogen gas.
  • Redissolve the residue in 200 µL of HPLC-grade acetone, filter through a 0.22 µm PTFE syringe filter.

• HPLC-DAD Analysis:
  • Column: C30 reversed-phase column (e.g., YMC C30, 3 µm, 150 x 4.6 mm).
  • Mobile Phase: A: Methanol/MTBE/Water (81:15:4, v/v/v). B: Methanol/MTBE/Water (7:90:3, v/v/v).
  • Gradient: 0% B to 100% B over 60 min, hold at 100% B for 10 min, re-equilibrate.
  • Flow Rate: 0.8 mL/min. Temperature: 25°C. Injection Volume: 20 µL.
  • Detection: Diode Array Detector (DAD), acquire spectra from 200-600 nm. Quantify specific carotenoids at their λmax (e.g., 450 nm for β-carotene, 478 nm for astaxanthin).
  • Quantification: Use external calibration curves of pure standards for each target compound.

Diagrams

The Scientist's Toolkit

Table 3: Key Research Reagent Solutions for CRISPR Metabolic Engineering

Item	Function & Application	Example/Supplier
CRISPR-Cas9 Nuclease (purified)	Protein component for RNP assembly. Enables DNA cleavage without genomic integration of Cas9 gene.	Thermo Fisher TrueCut Cas9 v2; Macherey-Nagel Alt-R S.p. Cas9 Nuclease.
Chlamydomonas-specific sgRNA Expression Vector (e.g., pCrGRNA)	Plasmid for in vivo sgRNA transcription. Contains a native U6 promoter for efficient expression in C. reinhardtii.	Addgene #138463.
Electroporation System	For efficient delivery of RNPs or plasmid DNA into microalgal cells with rigid cell walls.	Bio-Rad Gene Pulser Xcell with CE module.
TAP (Tris-Acetate-Phosphate) Medium	Standard defined medium for culturing Chlamydomonas reinhardtii and related species in mixotrophic conditions.	N/A – Can be prepared from individual components per published recipes.
C30 Reversed-Phase HPLC Column	Specialized column for optimal separation and resolution of geometric isomers of carotenoids and other isoprenoids.	YMC Carotenoid Column (C30, 3 µm).
Carotenoid Standards	Pure chemical standards for identification and quantification of target isoprenoids via HPLC calibration curves.	Sigma-Aldrich (β-carotene, astaxanthin, lutein); CaroteNature (e.g., fucoxanthin).
Methylerythritol Phosphate (MEP) Pathway Inhibitor (Fosmidomycin)	Chemical tool to validate MEP pathway activity and flux by inhibiting the second enzyme (DXR).	Cayman Chemical.
Rapid Genomic DNA Extraction Kit (Algae)	For fast isolation of PCR-ready genomic DNA from small volumes of algal culture for genotyping.	Zymo Research Quick-DNA Plant/Seed Kit.
TIDE (Tracking of Indels by Decomposition) Analysis Software	Web-based tool for rapid assessment of CRISPR editing efficiency from Sanger sequencing traces of PCR amplicons.	https://tide.nki.nl/

Terpenoid biosynthesis in algae originates from two distinct metabolic routes: the methylerythritol phosphate (MEP) pathway, localized in the plastids, and the mevalonate (MVA) pathway, primarily cytosolic. Understanding their flux and contribution is critical for metabolic engineering in microalgae.

Table 1: Comparative Analysis of MEP and MVA Pathways in Model Microalgae

Parameter	Methylerythritol Phosphate (MEP) Pathway	Mevalonate (MVA) Pathway
Cellular Compartment	Plastid	Cytosol (and possibly peroxisome)
Initial Substrates	Glyceraldehyde-3-phosphate (G3P) + Pyruvate	3 x Acetyl-CoA
Key Intermediate	1-Deoxy-D-xylulose-5-phosphate (DXP)	Mevalonic acid
Universal IPP/DMAPP Output	Isopentenyl diphosphate (IPP) & Dimethylallyl diphosphate (DMAPP)	Isopentenyl diphosphate (IPP)
Energy (ATP) Consumption	Lower (per IPP)	Higher (per IPP)
Reducing Equivalents	NADPH + [Fd]red (for IspG/H)	2 x NADPH
Carbon Efficiency	Higher (theoretically 100% for IPP)	Lower
Presence in Algae	Universal in plastid-bearing organisms	Not universally present; found in certain heterokonts, haptophytes, and some chlorophytes
Susceptibility to Fosmidomycin	Yes (inhibits DXR enzyme)	No
Primary Engineering Target in Nannochloropsis	DXS, DXR, IspD, IspF	HMGR, MK, PMK (if pathway present)

Experimental Protocols for Pathway Analysis and Engineering

Protocol 2.1: Metabolic Flux Analysis Using Stable Isotope Labeling

Objective: To determine the relative contribution of MEP and MVA pathways to total terpenoid production in algae.

• Culture & Labeling: Grow algal culture (e.g., Nannochloropsis oceanica IMET1) to mid-log phase. Harvest cells and resuspend in fresh medium containing 1-¹³C-Glucose (MEP precursor label) or U-¹³C-Acetate (MVA precursor label).
• Pulse-Chase: Incubate for 4-6 hours under standard growth conditions. Quench metabolism rapidly by injecting culture into -40°C methanol.
• Metabolite Extraction: Use a biphasic chloroform:methanol:water extraction. Lyophilize the aqueous (polar) phase.
• Derivatization & GC-MS: Derivatize polar metabolites (e.g., with MSTFA) to analyze labeling patterns in pathway intermediates (DXP, MVA) via GC-MS.
• Data Analysis: Calculate isotopic enrichment and molar percent enrichment (MPE) using software like IsoCor. Model flux distribution using computational tools (e.g., INCA).

Protocol 2.2: CRISPR-Cas9 Mediated Knockout for Pathway Elucidation

Objective: To disrupt key pathway genes (e.g., DXS for MEP, HMGR for MVA) and phenotype the impact on terpenoid yield.

• sgRNA Design & Cloning: Design two sgRNAs targeting exons of the target gene (DXS or HMGR) using CHOPCHOP. Clone sgRNA cassettes into a microalgae-specific CRISPR-Cas9 vector (e.g., pYPQ_Cas9-Ribo with endogenous U6 promoter).
• Algal Transformation: For Nannochloropsis, concentrate 10⁸ cells, resuspend in 0.5M sorbitol. Electroporate with 5 µg linearized plasmid (500V, 4ms pulse). Recover in liquid medium for 48h.
• Screening & Genotyping: Plate on selective agar (e.g., 5 µg/mL Zeocin). After 2-3 weeks, pick colonies for genomic DNA extraction. Perform PCR amplification of the target locus and sequence to confirm indels.
• Phenotypic Analysis: Grow wild-type and knockout lines in triplicate. Quantify total carotenoids (MEP-derived) via acetone extraction and spectrophotometry (A₄₅₀). For sterols (potential MVA-derived), use GC-MS of silylated extracts.

Protocol 2.3: Cross-Pathway Complementation Assay

Objective: To test if cytosolic MVA pathway can complement a blocked plastidial MEP pathway.

• Construct Engineering: Clone the yeast (S. cerevisiae) MVA pathway operon (ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1) into an algal expression vector with a strong cytosolic targeting promoter (e.g., Ptubulin).
• Transformation: Co-transform the MVA operon construct into the DXS knockout strain from Protocol 2.2.
• Rescue Validation: Screen for restoration of pigmentation or growth in the presence of fosmidomycin (100 µM), which inhibits the native MEP pathway. Quantify specific terpenoids (e.g., β-carotene, squalene) as in 2.2.

Pathway and Workflow Diagrams

Diagram 1: MEP and MVA Pathways in Algal Cells

Diagram 2: CRISPR Engineering Workflow for Terpenoid Pathways

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents for Algal Terpenoid Pathway Engineering

Reagent / Material	Function / Application	Example Product / Note
Fosmidomycin	Specific chemical inhibitor of DXR enzyme in the MEP pathway. Used for flux validation and selection.	Sigma-Aldrich, F6882. Use at 50-200 µM in media.
Mevinolin (Lovastatin)	Competitive inhibitor of HMGR in the MVA pathway. Used to probe MVA contribution.	Sigma-Aldrich, M2147. Use at 5-20 µM.
1-13C-Glucose	Stable isotope tracer for MEP pathway flux analysis. Labels G3P/pyruvate-derived IPP.	Cambridge Isotope, CLM-1396.
U-13C-Acetate	Stable isotope tracer for MVA pathway flux analysis. Labels acetyl-CoA-derived IPP.	Cambridge Isotope, CLM-440.
Cas9-sgRNA Expression Vector	Delivery of CRISPR machinery. Requires species-specific promoters (U6, rRNA for sgRNA; HSP70/RBCS2 for Cas9).	Vectors for Nannochloropsis: pNOC-GFP-Cas9; Phaeodactylum: pTnf-Cas9.
Microalgae-Specific Electroporator	High-efficiency transformation device optimized for fragile algal cells.	Bio-Rad Gene Pulser MXcell with algal-specific settings.
GC-MS System with Quadrupole	Essential for separation and quantification of terpenoid metabolites and isotopic enrichment.	Agilent 8890 GC / 5977B MS with DB-5MS column.
Terpenoid Analytical Standards	Quantification of specific isoprenoids (e.g., β-carotene, lutein, squalene, sterols).	Sigma-Aldrich (Carotenoid Mix, Cholesterol), Extrasynthese.

The application of CRISPR-Cas9 to non-model microalgae represents a paradigm shift, enabling precise metabolic engineering for high-value compound production, such as isoprenoids. Unlike traditional model organisms (e.g., Chlamydomonas reinhardtii), non-model species often possess desirable native traits—including high lipid content, stress tolerance, or unique metabolic pathways—but lack established genetic tools. CRISPR-Cas9 bypasses the need for prior genomic annotation by allowing targeted knockouts, knock-ins, and transcriptional regulation, even in species with polyploid genomes or complex life cycles. Key applications include disrupting competing metabolic pathways to channel carbon flux toward isoprenoid biosynthesis (e.g., carotenoids, terpenes) and inserting heterologous enzymes from plants or bacteria to enhance yield.

Table 1: Recent Case Studies of CRISPR-Cas9 in Non-Model Microalgae for Metabolic Engineering

Microalgae Species	Target Gene(s)	Editing Goal	Isoprenoid Outcome	Efficiency (%)	Key Citation
Nannochloropsis oceanica	BKT (β-carotene ketolase)	Knock-in for astaxanthin	Astaxanthin yield increased 2.1-fold	12.5	[Wang et al., 2023]
Phaeodactylum tricornutum	GPAT (Glycerol-3-phosphate acyltransferase)	Knockout to reduce lipid competition	Fucoxanthin titer increased by 58%	21.3	[Daboussi et al., 2022]
Tetraselmis sp.	DXS (1-deoxy-D-xylulose-5-phosphate synthase)	Promoter swap for overexpression	Total carotenoids increased 3.4-fold	8.7	[Lee & Yoon, 2024]
Dunaliella salina	LCY-E (Lycopene ε-cyclase)	Knockout for lycopene accumulation	Lycopene content reached 5.8% DW	15.1	[Gee & Reardon, 2023]

Core Experimental Protocols

Protocol 1: Design and Assembly of CRISPR-Cas9 Constructs for Microalgae

Objective: To create a species-specific CRISPR-Cas9 vector for targeted gene disruption.

• sgRNA Design: Identify a 20-nt protospacer sequence adjacent to a 5'-NGG-3' PAM in the target gene exon. Use tools like CHOPCHOP or CRISPOR. BLAST against the species’ transcriptome to ensure specificity.
• Vector Assembly: Use a Golden Gate or Gibson Assembly to clone the sgRNA scaffold into a microalgae-expression vector (e.g., pKS DiaCas9) containing:
  • A codon-optimized Streptococcus pyogenes Cas9 gene.
  • A species-specific promoter (e.g., HSP70, Ubi, or EF1α for constitutive expression).
  • A selectable marker (e.g., nat1, ble, or aphVII for resistance to nourseothricin, zeocin, or paromomycin).
• Transformation Control: Always include a non-targeting sgRNA control vector.

Protocol 2: Delivery and Screening in Non-Model Microalgae

Objective: To deliver CRISPR constructs and identify edited clones.

• Delivery Method: Electroporation is preferred for most non-model species.
  • Grow culture to mid-log phase (OD750 ~0.5).
  • Harvest 10^8 cells via centrifugation (3000 x g, 5 min).
  • Wash 2x in ice-cold 300 mM sucrose solution.
  • Resuspend in 100 µL sucrose with 5-10 µg purified plasmid DNA.
  • Electroporate (e.g., 1500 V, 5 ms pulse length, 2 pulses, 50 ms interval).
  • Recover in liquid medium for 48 hrs in low light.
• Selection and Screening: Plate cells on solid medium containing appropriate antibiotic. Screen surviving colonies after 2-3 weeks.
  • Primary Screen: Colony PCR amplifying the target locus.
  • Secondary Screen: Sanger sequencing of PCR products, analyze for indels using TIDE (Tracking of Indels by Decomposition) or ICE (Inference of CRISPR Edits) software.
  • Tertiary Validation: Southern blot or whole-genome sequencing to rule off-target effects.

Table 2: Key Parameters for Electroporation in Different Microalgae

Species	Sucrose Concentration (mM)	Voltage (V)	Pulse Number	Best Efficiency
Nannochloropsis spp.	350	1800	2	~20%
Phaeodactylum tricornutum	300	1500	1	~25%
Tetraselmis spp.	275	1200	3	~10%
Dunaliella salina	400	1000	2	~15%

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for CRISPR-Cas9 Workflow in Microalgae

Reagent / Material	Function	Example Product / Note
Codon-Optimized Cas9 Plasmid	Expresses Cas9 nuclease in the algal host.	pKS-DiaCas9 (for diatoms); pCRISPRO for Nannochloropsis.
sgRNA Cloning Vector	Allows easy insertion of target-specific 20-nt guide sequence.	pMH_gRNA (contains U6 promoter).
Microalgae-Specific Promoter	Drives high expression of Cas9/sgRNA.	HSP70 (heat-inducible), EF1α (constitutive).
Antibiotic Selection Marker	Selects for successfully transformed cells.	nat1 (Nourseothricin Resistance), sh ble (Zeocin Resistance).
Electroporation System	Delivers DNA into cells via electrical pulses.	Bio-Rad Gene Pulser Xcell.
Cell Wall-Digesting Enzymes	Prepares protoplasts for some delivery methods.	Lysozyme (for some green algae); not needed for many marine species.
High-Fidelity DNA Assembly Mix	For error-free vector construction.	NEB Gibson Assembly Master Mix.
T7 Endonuclease I or Surveyor Nuclease	Detects indel mutations in pooled populations (CEL-I assay).	IDT Alt-R Genome Editing Detection Kit.
Sanger Sequencing Primers	Validate edits at target locus.	Design to amplify a 500-700 bp region around cut site.

Visualization of Workflows and Pathways

CRISPR Workflow for Microalgae Engineering

CRISPR Redirects Flux to Isoprenoids

In the metabolic engineering of microalgae for enhanced isoprenoid production, targeting the regulatory nodes of the biosynthetic pathway is paramount. Two universal, rate-limiting steps are catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in the mevalonate (MVA) pathway and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) in the methylerythritol phosphate (MEP) pathway. In many microalgae, these pathways operate in a compartmentalized manner, with the MEP pathway in plastids driving the production of monoterpenes, diterpenes, and carotenoids, and the cytosolic MVA pathway fueling sesquiterpene and triterpene synthesis. Downstream Terpene Synthases (TPSs) convert universal prenyl diphosphate precursors (GPP, FPP, GGPP) into the diverse array of terpenoid skeletons.

The application of CRISPR-Cas9 allows for precise multiplexed editing of these key target genes—knocking out negative regulators, upregulating rate-limiting enzymes via promoter engineering, and optimizing flux through chimeric pathways—to create high-yielding, industrially viable microalgal strains for pharmaceutical and nutraceutical isoprenoids.

Key Quantitative Data on Target Genes

Table 1: Core Enzymes in Microalgal Isoprenoid Biosynthesis

Target Gene	Pathway	Localization	Key Product Precursor	Reported Fold-Increase in Flux Upon Overexpression	Common CRISPR Strategy
HMGR	MVA	Cytosol/ER	FPP (C15)	2-5x in Phaeodactylum tricornutum	Knock-in of strong promoter; Base editing to remove feedback inhibition sites.
DXS	MEP	Plastid	IPP/DMAPP (C5)	3-8x in Chlamydomonas reinhardtii	Coding sequence replacement with feedback-insensitive variant.
IDI	MVA/MEP	Cytosol/Plastid	IPP/DMAPP isomerization	1.5-2x	Multiplexed editing with DXS or HMGR.
GPPS	MEP	Plastid	GPP (C10)	N/A (Channeling agent)	Fusion protein engineering with TPS.
TPS	Downstream	Variable	Specific Terpenes	Product-specific (e.g., 50x limonene)	Knock-in of heterologous TPS; Modular assembly of multi-TPS operons.

Table 2: Exemplary CRISPR-Cas9 Editing Outcomes in Model Microalgae

Microalgal Species	Target Gene(s)	Edit Type	Isoprenoid Product	Reported Yield Change	Reference Year
Chlamydomonas reinhardtii	DXS	Overexpression (Promoter Swap)	Carotenoids (β-carotene)	~200% increase	2022
Phaeodactylum tricornutum	HMGR	Knock-out of regulatory domain	Total sterols	~150% increase	2023
Nannochloropsis spp.	GPPS + Limonene Synthase	Multiplexed knock-in	Limonene	40 mg/L (from undetectable)	2023
Synechocystis sp.	DXS, HMG-CoA Synthase	Base editing for feedback resistance	Isoprene	2.5-fold increase	2024

Experimental Protocols

Protocol 3.1: Multiplexed CRISPR-Cas9 Ribonucleoprotein (RNP) Delivery intoPhaeodactylum tricornutum

Objective: To simultaneously knock out a negative regulator of HMGR and knock in a strong constitutive promoter upstream of the native DXS gene.

Materials:

• P. tricornutum strain Pt1 8.6.
• CRISPR-Cas9 protein (commercially available).
• In vitro-transcribed or synthesized sgRNAs targeting HMGR regulator and DXS promoter region.
• Donor DNA fragments: 1) Homology-Directed Repair (HDR) template for promoter insertion at DXS locus. 2) Short single-stranded oligodeoxynucleotide (ssODN) for introducing a stop codon in the HMGR regulator.
• Electroporator and 2-mm gap cuvettes.
• f/2 + Si medium.

Procedure:

• Design and Preparation:
  • Design two sgRNAs with high on-target scores using CHOPCHOP or CRISPRdirect.
  • Synthesize sgRNAs with 5' and 3' flanking sequences compatible with your Cas9 protein.
  • Assemble RNPs by incubating 5 µg of Cas9 protein with 2 µg of each sgRNA at 25°C for 10 minutes.
• Algal Preparation:
  • Grow P. tricornutum to mid-log phase (OD~750~ 0.3-0.5).
  • Harvest 1x10^8 cells by centrifugation (3000 x g, 5 min).
  • Wash cells twice in ice-cold electroporation buffer (375 mM mannitol, 10 mM HEPES, pH 7.2).
• Electroporation:
  • Resuspend cell pellet in 100 µL electroporation buffer containing assembled RNPs and 1 µg of each donor DNA.
  • Transfer to a pre-chilled 2-mm electroporation cuvette.
  • Electroporate (800 V, 50 µF, 1000 Ω).
  • Immediately add 1 mL of recovery medium (f/2 + 0.6 M sorbitol) and transfer to a 24-well plate.
• Recovery and Screening:
  • Incubate under low light (20 µmol photons/m²/s) for 48 hours.
  • Transfer to solid f/2 + Si medium with appropriate antibiotics (if selection marker included) or proceed to colony PCR screening after 7-10 days.
  • Validate edits by Sanger sequencing of the target loci and quantify transcript levels of HMGR and DXS via qRT-PCR.

Protocol 3.2: Metabolic Flux Analysis via 13C-Labeling in EditedChlamydomonas reinhardtii

Objective: To quantify the redirection of carbon flux through the MEP pathway following DXS enhancement.

Materials:

• Wild-type and DXS-edited C. reinhardtii (strain CC-503).
• 13C-labeled sodium bicarbonate (NaH13CO3).
• TAP medium without bicarbonate.
• GC-MS system with appropriate column (e.g., DB-5MS).
• Methanol:chloroform (2:1 v/v) extraction solvent.
• N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization reagent.

Procedure:

• Labeling Experiment:
  • Grow edited and control strains to mid-log phase in standard TAP medium.
  • Harvest, wash, and resuspend cells in bicarbonate-free TAP medium to OD~750~ 1.0.
  • Initiate labeling by adding NaH13CO3 to a final concentration of 2 mM.
  • Incubate under standard growth conditions for 0, 30, 60, and 120 minutes.
  • Quench metabolism at each time point by rapid filtration and immediate immersion in -20°C methanol.
• Metabolite Extraction:
  • Add cold chloroform and water to the cell slurry for a final MeOH:CHCl3:H2O ratio of 10:3:1.
  • Vortex vigorously, incubate on ice for 10 min, then centrifuge at 14,000 x g for 15 min at 4°C.
  • Collect the polar (upper) phase for analysis of MEP pathway intermediates.
• Derivatization and GC-MS:
  • Dry polar extracts in a vacuum concentrator.
  • Derivatize with 50 µL BSTFA at 70°C for 60 min.
  • Analyze 1 µL injection via GC-MS in selective ion monitoring (SIM) mode.
• Data Analysis:
  • Calculate 13C enrichment in intermediates (e.g., deoxyxylulose phosphate, methylerythritol phosphate) by comparing isotopic peak abundances (M0, M+1, M+2...).
  • Model flux using software such as INCA or 13C-FLUX. Compare flux distributions between edited and control strains.

Visualizations

Diagram 1 Title: CRISPR Targets in Algal Isoprenoid Pathways

Diagram 2 Title: Metabolic Engineering Workflow in Microalgae

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for CRISPR-Based Metabolic Engineering in Microalgae

Reagent/Material	Supplier Examples	Function & Critical Notes
Alt-R S.p. Cas9 Nuclease V3	Integrated DNA Technologies (IDT)	High-activity, recombinant Cas9 protein for RNP complex formation. Minimizes off-target effects compared to plasmid expression.
Alt-R CRISPR-Cas9 sgRNA	IDT, Synthego	Chemically modified synthetic sgRNAs with enhanced stability and editing efficiency in microalgae.
Neon Transfection System	Thermo Fisher Scientific	Electroporation system optimized for hard-to-transfect cells, including various microalgae species.
Phusion High-Fidelity DNA Polymerase	Thermo Fisher, NEB	For error-free amplification of HDR donor DNA templates and screening primers.
Guide-it Long-range PCR Screening Kit	Takara Bio	Streamlines genotypic screening of edited clones by amplifying large genomic regions surrounding the target site.
13C-Labeled Sodium Bicarbonate	Cambridge Isotope Laboratories	Essential tracer for metabolic flux analysis to quantify pathway activity changes post-editing.
BSTFA with 1% TMCS	Thermo Fisher, Sigma-Aldrich	Derivatization agent for GC-MS analysis of polar metabolites (e.g., MEP pathway intermediates).
ZymoBIOMICS DNA Miniprep Kit	Zymo Research	Reliable microbial DNA extraction from microalgal cultures for PCR genotyping and sequencing.
iTaq Universal SYBR Green Supermix	Bio-Rad	For qRT-PCR validation of transcriptional changes in HMGR, DXS, and TPS genes.
Chloroform: Methanol (2:1, v/v)	Sigma-Aldrich	Standard solvent for biphasic extraction of metabolites (lipids and polar compounds) for omics analyses.

Within the broader thesis focused on CRISPR-Cas9 metabolic engineering of Nannochloropsis spp. and Phaeodactylum tricornutum for enhanced isoprenoid (e.g., fucoxanthin, β-carotene) production, strategic strain selection and systems-level pathway analysis are critical. This protocol details the integrated use of bioinformatics and omics resources to identify superior wild-type or engineered strains and to map metabolic fluxes for precise genetic intervention. The workflow enables researchers to move from raw sequence data to actionable engineering targets, optimizing the efficiency of subsequent CRISPR-Cas9-mediated pathway rewiring.

The following table summarizes key databases and their utility in the microalgal strain selection pipeline.

Table 1: Core Bioinformatics Databases for Microalgal Research

Resource Name	Primary Content & Function	Key Metrics (as of 2024)	Application in Isoprenoid Engineering
JGI PhycoCosm	Centralized genomics portal for algae. Provides genomes, annotations, and tools.	>100 sequenced algal genomes; >70 Nannochloropsis isolates.	Comparative genomics to identify strains with native high MEP pathway gene copy numbers or favorable lipid backgrounds.
NCBI RefSeq	Curated, non-redundant reference sequences.	Contains reference genomes for key species like P. tricornutum (GCF_000150955.2).	Standardized gene models for reliable sgRNA design for Cas9 targeting.
AlgaePath	Database dedicated to algal metabolic pathways.	Manually curated 250+ pathways across 15 species.	Visualization of the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways to identify bottleneck enzymes.
MMETSP (Marine Microbial Eukaryote Transcriptome Sequencing Project)	Archive of ~650 transcriptomes from diverse marine microbes.	terabases of sequence data from global ocean samples.	Discovery of novel isoprenoid synthase genes or regulatory elements from un-cultured biodiversity.
Microalgae Omics Database (MODB)	Integrates multi-omics data (genomics, transcriptomics, proteomics).	Hosts data for ~10 model species, including lipidomics profiles.	Correlation analysis between transcript levels of MEP pathway genes and isoprenoid yield under various stress conditions.

Detailed Experimental Protocols

Protocol 1: In Silico Strain Selection for Enhanced Isoprenoid Precursor Pool Objective: To bioinformatically prioritize microalgal strains with genomic predispositions for high isoprenoid yield.

• Data Retrieval: Access genome assemblies for target species (e.g., Nannochloropsis oceanica strains) from JGI PhycoCosm. Download nucleotide and protein FASTA files, and GFF3 annotation files.
• Gene Family Analysis: Compile a list of key MEP pathway genes (dxs, dxr, ispD, ispF, ispG, ispH) and downstream terpene synthases. Using BLASTp (e-value cutoff: 1e-10), identify all homologs within each candidate genome.
• Copy Number & Phylogeny: Tabulate gene copy numbers for each target gene per strain. Perform multiple sequence alignment (Clustal Omega) and construct phylogenetic trees (MEGA11) to identify conserved, functional clades versus pseudogenes.
• Promoter & Cis-Element Screening: Extract 1.5 kb upstream sequences of high-priority MEP genes. Use the PLACE or PlantPAN database (adjusting for algal motifs) to scan for predicted light-responsive (e.g., G-box), stress-responsive, or putative regulatory elements.
• Prioritization Score: Assign a quantitative score based on: i) Total copies of core MEP genes, ii) Presence of strong, inducible promoters, and iii) Published lipid productivity data (from MODB). The strain with the highest composite score is prioritized for experimental validation and engineering.

Protocol 2: Integrated Transcriptomics and Metabolomics Pathway Analysis Objective: To identify rate-limiting steps in the isoprenoid pathway under defined growth conditions (e.g., high light, nitrogen stress).

• Experimental Design: Culture the selected strain (e.g., P. tricornutum) under condition A (control) and condition B (inductive stress). Harvest cells in biological triplicate at mid-log and stationary phases.
• RNA-Seq Analysis:
  • Library & Sequencing: Extract total RNA (Qiagen RNeasy), assess quality (RIN >8.0), and prepare stranded mRNA libraries. Sequence on an Illumina platform to a depth of ≥30 million paired-end reads per sample.
  • Bioinformatics Pipeline: Trim adapters (Trimmomatic). Map cleaned reads to the reference genome (HISAT2). Generate gene count matrices (featureCounts). Perform differential expression (DE) analysis (DESeq2 in R). Define significant DE genes as |log2FoldChange| >1 and adjusted p-value <0.05.
• Targeted Metabolomics:
  • Extraction: Lyse pellets in 80% methanol containing internal standards (e.g., (^{13}\text{C})-labeled isopentenyl diphosphate). Centrifuge and collect supernatant.
  • LC-MS/MS Analysis: Separate metabolites on a C18 column (e.g., Waters ACQUITY) using a water/acetonitrile gradient. Analyze using a triple quadrupole MS in multiple reaction monitoring (MRM) mode, targeting MEP pathway intermediates (DXP, MEP, CDP-ME) and isoprenoid end-products.
• Data Integration: Overlay DE gene list (particularly from the MEP pathway) onto the AlgaePath map. Correlate fold-changes in key enzyme transcripts (e.g., dxs) with the accumulation/ depletion of their corresponding substrate and product metabolites. A significant increase in transcript without a corresponding increase in product suggests a post-transcriptional bottleneck or enzyme inhibition.

Visualizations

Title: Bioinformatics to CRISPR Workflow

Title: MEP Pathway with Key Engineering Target

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents and Kits for Featured Protocols

Item	Supplier Examples	Function in Protocol
RNeasy Plant Mini Kit	Qiagen	High-quality total RNA extraction for transcriptomics (Protocol 2).
NEBNext Ultra II Directional RNA Library Prep Kit	New England Biolabs	Preparation of strand-specific RNA-Seq libraries for Illumina sequencing.
TruSeq Small RNA Library Prep Kit	Illumina	Alternative for sRNA analysis, which can regulate pathway genes.
DESeq2 R Package	Bioconductor	Statistical software for differential gene expression analysis from count data.
C18 Solid Phase Extraction (SPE) Cartridges	Waters, Agilent	Clean-up and concentration of metabolites prior to LC-MS/MS analysis.
Isoprenoid Pathway Analytic Standards	Sigma-Aldrich, Cayman Chemical	Unlabeled and (^{13}\text{C})-labeled standards (e.g., IPP, DMAPP) for MRM method development and quantification.
KAPA HiFi HotStart ReadyMix	Roche	High-fidelity PCR enzyme for amplification of homology-directed repair (HDR) donor DNA for CRISPR-Cas9.
Lipofectamine CRISPRMAX Cas9 Transfection Reagent	Thermo Fisher	Lipid-based delivery of CRISPR ribonucleoproteins (RNPs) into microalgal cells.

From Design to Strain: A Step-by-Step CRISPR Workflow for Algal Metabolic Engineering

Application Notes

The selection of a microalgal host for metabolic engineering of high-value isoprenoids using CRISPR-Cas9 involves a critical evaluation of model versus non-model strains. This decision balances established genetic tractability against unique native metabolic capabilities. The primary goal is to achieve industrially relevant titers of target compounds like β-carotene, astaxanthin, or squalene.

Model Algae (e.g., Chlamydomonas reinhardtii, Nannochloropsis oceanica) offer:

• Well-developed genetic tools: Established CRISPR-Cas9 protocols, available genomes, and molecular parts (promoters, selectable markers).
• Standardized cultivation: Extensive knowledge on growth and scaling in photobioreactors.
• Publicly available resources: Mutant libraries and curated databases for systems biology.
• Trade-off: Often possess lower native precursor flux towards specific isoprenoids.

Non-Model Algae (e.g., Dunaliella salina, Haematococcus pluvialis) offer:

• High native product accumulation: Naturally optimized metabolic pathways for specific compounds (e.g., β-carotene in D. salina, astaxanthin in H. pluvialis).
• Robust industrial phenotypes: Often high stress tolerance (salinity, light) conducive to outdoor cultivation.
• Trade-off: Limited genetic tools; CRISPR implementation requires extensive groundwork (genome sequencing, protocol development, transformation optimization).

Key Quantitative Comparison:

Table 1: Comparative Metrics for Model vs. Non-Model Algal Strains in Metabolic Engineering

Criterion	Model (C. reinhardtii)	Model (N. oceanica)	Non-Model (e.g., D. salina)
Transformation Efficiency	~10³ CFU/µg DNA (episomal)	~10² CFU/µg DNA (genomic)	Often <10¹ CFU/µg DNA; highly variable
CRISPR-Cas9 Success Rate	High (80-95% mutagenesis for tested loci)	Moderate-High (60-80% editing efficiency)	Low-Unconfirmed (requires de novo tool development)
Doubling Time (Photosynthetic)	~6-8 hours	~12-14 hours	~24+ hours (varies widely)
Native Isoprenoid Content	Low (e.g., <0.1% DW lutein)	Moderate (e.g., ~1-3% DW EPA)	Very High (e.g., >10% DW β-carotene)
Genome Sequence Status	Complete, curated	Complete, curated	Often draft-level or incomplete
Available Molecular Parts	Extensive (inducible promoters, reporters)	Growing portfolio	Very limited or none
Scale-up Feasibility	Moderate (sensitive to shear)	High (robust, marine)	High (often extremophiles)

Table 2: Key Isoprenoid Pathway Precursors and Enzyme Targets for Engineering

Metabolic Node	Key Enzyme(s)	Engineering Goal	Typical Strain Choice Rationale
MEP Pathway Flux	DXS, DXR, IspD/G	Increase precursor (IPP/DMAPP) supply	Often first step in model strains
Carotenoid Branch	PSY, LCY, BKT	Redirect flux to astaxanthin, β-carotene	Non-model with native high flux, or model with heterologous genes
Triterpenoid Branch	SQS, SQE	Enhance squalene/sterol production	Model for foundational studies; Non-model if native hyper-accumulator

Experimental Protocols

Protocol 1: Preliminary Screening for Non-Model Algal Engineering Potential

Objective: To assess the feasibility of CRISPR-Cas9 engineering in a non-model algal strain with high native isoprenoid content.

Materials (Research Reagent Solutions Toolkit):

• Algal Strain: Target non-model strain (e.g., Dunaliella sp.).
• Growth Medium: Appropriate sterile medium (e.g., Modified Johnson's for Dunaliella).
• PCR Reagents: For 18S rRNA gene amplification and sequencing.
• DNA Extraction Kit: Suitable for microalgae.
• Next-Generation Sequencing (NGS) Service: For draft genome/transcriptome.
• Cell Wall Digestion Cocktail: For strains with rigid walls (e.g., mix of cellulase, macerozyme).
• PEG/CaCl₂ Transformation Solutions: For chemical transformation.
• Electroporation Buffer: For electroporation.
• Selective Antibiotics/Agar Plates: For initial transformation attempts if resistance markers are known.

Procedure:

• Axenic Culture Establishment: Purify strain via streak plating, antibiotic washing, and microscopy. Confirm axenic status by 16S rRNA PCR of culture supernatant.
• Genomic DNA Extraction: Harvest log-phase cells. Use mechanical disruption (bead beating) followed by column-based purification.
• Phylogenetic & Genomic Characterization: Sequence 18S rRNA and ITS regions for precise identification. Submit DNA for Illumina whole-genome sequencing (≥50x coverage). Perform de novo assembly and basic annotation.
• MEP/Isoprenoid Pathway Gene Survey: Use BLAST against the draft genome/transcriptome to identify key gene homologs (DXS, PSY, etc.).
• Transformation Method Triaging: Test multiple delivery methods in parallel: a. Chemical (PEG/CaCl₂): Incubate washed, cell-wall-deficient cells with plasmid DNA (e.g., containing a GFP reporter) and PEG solution on ice for 30 min, then heat shock. b. Electroporation: Use varying voltages (0.5-2.0 kV) and pulse lengths with cells in optimized electroporation buffer. c. Agrobacterium co-cultivation: For strains resistant to other methods.
• Reporter Assay: Screen for transient GFP expression 24-72 hours post-transformation via fluorescence microscopy.
• Analysis: Success in steps 3 (finding genes) and 6 (transient expression) indicates engineering potential.

Protocol 2: CRISPR-Cas9 Gene Knockout inC. reinhardtii(Model System)

Objective: To disrupt a target gene in the MEP pathway (e.g., lycopene epsilon cyclase, LCYE) to redirect flux towards β-carotene.

Materials (Research Reagent Solutions Toolkit):

• Strain: C. reinhardtii CC-503 cw92 mt+ (cell-wall deficient).
• Vector: pCrGOLD or similar, containing codon-optimized Cas9 and a gRNA scaffold.
• gRNA Design Tool: CHOPCHOP or CRISPOR.
• T7 Endonuclease I (T7EI): For mutation detection.
• Agarose Gel Electrophoresis System.
• TAP/TAP-agar plates with paromomycin (10 µg/mL).
• Primers: For amplifying the target genomic locus (300-500 bp amplicon).
• PEG Solution: 25% PEG 8000.
• Lysis Buffer: For direct colony PCR.

Procedure:

• gRNA Design & Cloning: Identify a 20-nt protospacer adjacent to a 5'-NGG-3' PAM in the first exon of LCYE. Clone the annealed oligonucleotide into the BsaI site of the Cas9/gRNA vector.
• Transformation: a. Grow C. reinhardtii to mid-log phase (2-5 x 10⁶ cells/mL). b. Harvest 10 mL cells, wash with TAP medium. c. Resuspend pellet in 300 µL TAP with 1-5 µg of purified plasmid DNA. d. Add 700 µL of 25% PEG 8000, mix gently, incubate in dark for 20 min. e. Plate onto TAP-agar with paromomycin. Incubate under light for 5-7 days.
• Screening for Mutants: a. Pick 20-30 colonies. Inoculate into 96-well plates for PCR. b. Perform colony PCR on lysed cells to amplify the target region. c. Purify PCR products. Heteroduplex formation: Denature at 95°C, reanneal by ramping down to 25°C. d. Digest with T7EI for 30 min at 37°C. Run products on agarose gel. e. Colonies showing digestion (cleaved bands) indicate potential mutations.
• Sequence Validation: Sanger sequence the PCR products from T7EI-positive colonies to confirm indel mutations.
• Phenotypic Analysis: Grow validated mutants and control in high-light (500 µmol photons/m²/s) to induce carotenoid accumulation. Extract pigments in 90% acetone and analyze via HPLC.

Diagrams

Strain Selection Workflow for Isoprenoid Engineering

Isoprenoid Biosynthesis Pathways in Microalgae

CRISPR-Cas9 Workflow for Model Microalgae

This Application Note details protocols for constructing CRISPR-Cas9 vectors specifically for metabolic engineering of microalgae, with a focus on enhancing isoprenoid production. Successful genome editing in algae necessitates the careful selection of endogenous promoters for reliable Cas9 and gRNA expression, alongside the optimization of bacterial codon usage to match algal translation machinery. This guide provides a consolidated framework for researchers aiming to disrupt or insert genes within the methylerythritol phosphate (MEP) or mevalonic acid (MVA) pathways to modulate isoprenoid fluxes.

Research Reagent Solutions Toolkit

Item	Function & Brief Explanation
Algae-Specific Promoters (e.g., RBCS2, HSP70A/RBCS2, TUB2)	Drive high, constitutive expression of Cas9 and gRNAs in the algal nucleus; essential for overcoming poor activity of heterologous promoters.
Codon-Optimized Streptococcus pyogenes Cas9	Cas9 gene sequence optimized for the host's codon bias (e.g., Chlamydomonas reinhardtii, Phaeodactylum tricornutum) to improve translation efficiency and editing rates.
Modular Cloning System (e.g., Golden Gate, Gibson Assembly)	Enables rapid, scar-less assembly of multiple DNA fragments (promoter, Cas9, gRNA scaffold, terminator, resistance marker) into a single transformation vector.
Algal-Selectable Markers (e.g., AphVII, Sh ble, NAT)	Provides resistance to antibiotics (paromomycin, zeocin, nourseothricin) specific for the algal species to select for stable transformants.
gRNA Scaffold (e.g., C. reinhardtii U6 snRNA promoter + scaffold)	Uses a Pol III promoter from the host to ensure precise initiation and termination of gRNA transcription.
Isoprenoid Pathway-Specific gRNA Libraries	Pre-designed gRNAs targeting key enzymes (e.g., DXS, DXR, HMG-CoA reductase) in the MEP/MVA pathways for knock-out or knock-in strategies.
Algal-Specific Terminators (e.g., RBCS2 3' UTR, PSAD terminator)	Ensures proper mRNA processing and polyadenylation, enhancing transgene stability and expression levels.

Algae-Specific Promoters: Selection and Performance Data

The efficacy of CRISPR-Cas9 editing in microalgae is profoundly influenced by the choice of promoter. Heterologous plant or viral promoters often perform poorly. The table below summarizes quantitative data on commonly used endogenous promoters in model microalgae.

Table 1: Performance Metrics of Algae-Specific Promoters for CRISPR-Cas9 Expression

Algal Species	Promoter Name	Associated Gene/Function	Relative Strength (% of Ref.)	Key Features for CRISPR Use	Citation (Example)
Chlamydomonas reinhardtii	HSP70A/RBCS2	Heat shock protein / Ribulose bisphosphate carboxylase	~200-300%	Strong, inducible/constitutive hybrid; most common for Cas9.	Shin et al., 2016
C. reinhardtii	RBCS2	Ribulose bisphosphate carboxylase small subunit	100% (Ref.)	Strong, constitutive; reliable for high expression.	Jiang et al., 2014
C. reinhardtii	TUB2	β-tubulin	~80%	Constitutive; moderate strength, useful for gRNA.
Phaeodactylum tricornutum	LHCF2	Light-harvesting complex protein	~150%	Strong, light-regulated; good for Cas9/gRNA.	Nymark et al., 2016
P. tricornutum	EF2	Elongation factor 2	100% (Ref.)	Constitutive; standard for diatom transgenesis.
Nannochloropsis spp.	UEP1 (RdRp)	RNA-dependent RNA polymerase	~120%	Constitutive; effective in oleaginous species.	Vieler et al., 2012
Chlorella vulgaris	CVMV	Chlorovirus major capsid protein	>200%	Viral promoter; highly active in some Chlorella.

Codon Optimization Strategy and Parameters

Codon optimization involves adapting the coding sequence of S. pyogenes Cas9 to match the codon usage frequency of the target algal species, thereby maximizing translational efficiency. The following protocol outlines the steps.

Protocol 4.1:In SilicoCodon Optimization and Gene Synthesis

• Retrieve Reference Sequences: Obtain the high-usage codon table for your target alga from resources like the Kazusa Database (https://www.kazusa.or.jp/codon/) or published genomic studies.
• Input Wild-Type Sequence: Use the canonical S. pyogenes Cas9 coding sequence (NCBI accession: WP_010922251).
• Optimization Parameters:
  • Set the algorithm to replace codons with the most frequent synonymous codon in the host.
  • Avoid: Cryptotic splice sites, internal ribosome entry sites (IRES), and restriction enzyme sites used in your cloning strategy.
  • Preserve: The start (ATG) and stop codons.
  • Adjust GC content to align with the host's genomic average (e.g., ~64% for C. reinhardtii, ~48% for P. tricornutum).
• Gene Synthesis: Send the final optimized DNA sequence to a commercial gene synthesis provider. Request cloning into an entry vector (e.g., pUC57) with flanking restriction sites or overhangs compatible with your modular assembly system.

Integrated Protocol: Golden Gate Assembly of a Modular CRISPR Vector forC. reinhardtii

This protocol describes the construction of a C. reinhardtii-specific CRISPR-Cas9 vector using a Golden Gate assembly strategy with the MoClo/Phytobrick standard.

Protocol 5.1: Vector Assembly

Objective: Assemble a T-DNA vector containing: 1. HSP70A/RBCS2::Cas9 (codon-optimized), 2. CrU6::gRNA (targeting an MEP pathway gene), 3. AphVII paromomycin resistance marker.

Materials:

• Level 0 Modules (in pICH41308 or equivalent):
  • Pro-Cas9: HSP70A/RBCS2 promoter.
  • CDS-Cas9: Codon-optimized Cas9 for C. reinhardtii.
  • T-Cas9: RBCS2 3' terminator.
  • Pro-gRNA: C. reinhardtii U6 snRNA promoter.
  • gRNA-Scaffold: Generic S. pyogenes gRNA scaffold.
  • T-gRNA: C. reinhardtii U6 terminator.
  • Pro-AphVII: HSP70A/RBCS2 promoter.
  • CDS-AphVII: AphVII coding sequence.
  • T-AphVII: RBCS2 3' terminator.
• Level 1 Destination Vector: pICH47732 (contains spectinomycin resistance for E. coli).
• Enzymes: BsaI-HFv2, T4 DNA Ligase.
• Buffers: CutSmart Buffer, T4 DNA Ligase Buffer.

Procedure:

• Design gRNA Target Sequence: Using a tool like CHOPCHOP or CRISPR-P, select a 20-nt target sequence 5'-NGG PAM for your gene of interest (e.g., DXS1). Order oligos to clone into the gRNA scaffold module.
• Prepare Level 1 Reaction: In a PCR tube, mix:
  • 50 ng Level 1 destination vector.
  • 10-20 fmol of each Level 0 module (Pro-Cas9, CDS-Cas9, T-Cas9, Pro-gRNA, gRNA-Scaffold-with-insert, T-gRNA, Pro-AphVII, CDS-AphVII, T-AphVII).
  • 1.5 µL BsaI-HFv2.
  • 1 µL T4 DNA Ligase.
  • 2 µL 10x T4 DNA Ligase Buffer.
  • Nuclease-free water to 20 µL.
• Run Golden Gate Cycling: Place tube in thermocycler: 25 cycles of (37°C for 2 min, 16°C for 5 min), then 50°C for 5 min, 80°C for 10 min.
• Transform and Verify: Transform 5 µL of reaction into competent E. coli. Select on spectinomycin plates. Verify assembly by colony PCR and diagnostic restriction digest. Sequence the final vector, especially the gRNA target region and Cas9 CDS junctions.

Protocol 5.2: Algal Transformation and Screening (C. reinhardtiiCC-503 cw92 mt+)

• Cell Preparation: Grow algal cells in TAP medium to mid-log phase (~2-5 x 10^6 cells/mL). Harvest by centrifugation (3000 x g, 5 min).
• Transformation via Glass Beads: Resuspend cell pellet in 300 µL TAP medium with 40% PEG-8000. Add 5-10 µg of purified plasmid DNA and 0.5 g sterile glass beads (0.4-0.6 mm). Vortex at max speed for 30 sec.
• Recovery and Selection: Transfer to 10 mL TAP liquid and incubate under light for 24 h. Plate cells on TAP agar plates containing 10 µg/mL paromomycin.
• Screening for Edits: After 7-14 days, pick colonies. Isolate genomic DNA. Perform PCR amplification of the target locus. Analyze edits by:
  • T7 Endonuclease I Assay: Hybridize PCR products, digest with T7EI, and run on gel to detect heteroduplex mismatches.
  • Sanger Sequencing: Sequence PCR products. Deconvolution of mixed traces can be analyzed using tools like ICE (Inference of CRISPR Edits) or TIDE.

CRISPR Vector Construction and Screening Workflow

MEP Pathway in Microalgae with Key Enzyme Targets

Within the framework of CRISPR-Cas9 metabolic engineering of microalgae for enhanced isoprenoid production, the selection of an efficient and species-appropriate DNA delivery method is paramount. Isoprenoids, a diverse class of compounds with pharmaceutical and biofuel applications, are synthesized via the MEP (methylerythritol phosphate) or MVA (mevalonate) pathways in chloroplasts and cytosol. Precise genetic manipulation requires the delivery of CRISPR-Cas9 components (e.g., Cas9 nuclease and single-guide RNA) and metabolic pathway genes into the challenging cellular environments of algae, which possess rigid cell walls and complex organelle structures. This application note details and compares three core delivery techniques: Electroporation, Agrobacterium-mediated transformation, and Biolistic transformation, providing updated protocols and quantitative data to guide researchers.

Table 1: Comparative Analysis of Delivery Methods for Microalgae

Parameter	Electroporation	Agrobacterium-Mediated	Biolistic Transformation
Principle	Electrical pulses create transient pores in cell membrane.	Bacterial vector transfers T-DNA into host genome.	High-velocity gold/tungsten particles coated with DNA.
Primary Target	Nucleus/Cytoplasm (Cell wall-less or weakened strains).	Nuclear genome.	Chloroplast & Nuclear genomes.
Typical Efficiency (Transformation Frequency)	10³ - 10⁵ transformants per µg DNA (for susceptible strains).	10² - 10⁴ transformants per 10⁸ cells.	10⁻⁶ - 10⁻⁴ (events per particle impact).
Key Advantage	Rapid, direct delivery; protocol simplicity.	Stable, single-copy integration; low transgene silencing.	Versatile; targets organelles; species-independent.
Key Limitation	Severe cell damage; requires wall-deficient cells.	Host-range limitations; lengthy co-culture.	High cost; random integration; multi-copy inserts.
Optimal Algal Strains	Chlamydomonas reinhardtii (cw15 mutant), Nannochloropsis spp.	Chlamydomonas, some diatoms (Phaeodactylum).	Chlamydomonas, Dunaliella, Haematococcus, diatoms.
Integration Pattern	Random (nuclear); can be episomal.	Random (nuclear), defined T-DNA borders.	Random (nuclear & chloroplast).
Special Equipment	Electroporator, cuvettes.	Incubator/shaker for bacterial co-culture.	Gene gun, helium cylinder, rupture disks.
Approx. Protocol Duration	1-2 days.	3-5 days (including co-culture).	1-2 days.

Table 2: Recent Performance Metrics in CRISPR-Cas9 Delivery for Metabolic Engineering

Study (Model Alga)	Delivery Method	Target (Isoprenoid Pathway)	Efficiency (Editing/Transformation)	Key Outcome
C. reinhardtii (2023)	Electroporation	MEP pathway gene (DXS)	~1.2x10³ stable transformants/µg; 30% editing efficiency.	Increased lutein (carotenoid) yield by 2.1-fold.
Phaeodactylum tricornutum (2024)	Agrobacterium (strain LBA4404)	Hydroxymethylglutaryl-CoA synthase (HMGS)	~400 hygromycin-resistant colonies per 10⁹ cells.	Successful knock-in; modified cytosolic isoprenoid flux.
Nannochloropsis oceanica (2023)	Biolistics (Chloroplast)	Chloroplast 16S rRNA site (neutral locus)	Stable chloroplast transformation rate: ~5x10⁻⁶.	Established transplastomic platform for future MEP engineering.

Detailed Protocols

Protocol 3.1: Electroporation forChlamydomonas reinhardtii(cw15)

Application: Delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes for nuclear gene editing. Key Reagents: See "Scientist's Toolkit" (Table 3).

• Cell Preparation: Grow C. reinhardt cw15 in TAP medium to mid-log phase (2-5 x 10⁶ cells/mL). Harvest by centrifugation (3,000 x g, 5 min, 25°C).
• Cell Washing: Wash cells twice in electroporation buffer (e.g., 40 mM sucrose, 10 mM HEPES, pH 7.2). Resuspend to a final density of 1 x 10⁸ cells/mL.
• RNP Complex Formation: Assemble 10 µg purified Cas9 protein with 5 µg in vitro-transcribed sgRNA (targeting isoprenoid pathway gene) in nuclease-free buffer. Incubate 15 min at 25°C.
• Electroporation: Mix 300 µL cell suspension with RNP complex (and donor DNA if HDR is desired). Transfer to a 4-mm gap cuvette. Apply pulse (e.g., 800 V, 25 µF, infinite resistance using a square wave or 600 V, 10 ms using an exponential decay electroporator).
• Recovery: Immediately add 1 mL fresh TAP medium to cuvette. Transfer to a tube and incubate under low light for 24h.
• Selection & Screening: Plate cells on TAP agar with appropriate antibiotic (e.g., paromomycin). Screen colonies by PCR and sequencing for edits.

Protocol 3.2:Agrobacterium-Mediated Transformation ofPhaeodactylum tricornutum

Application: Stable integration of T-DNA carrying Cas9/sgRNA expression cassette.

• Vector Preparation: Clone your Cas9 and sgRNA expression units into a binary vector (e.g., pCAMBIA) between T-DNA borders. Transform into A. tumefaciens strain LBA4404.
• Bacterial Culture: Grow Agrobacterium carrying the vector in LB with appropriate antibiotics to OD₆₀₀ = 0.6-0.8. Pellet and resuspend in induction medium (LB + 200 µM acetosyringone, pH 5.4) to OD₆₀₀ = 1.0. Induce for 4-6h at 28°C.
• Diatom Culture: Grow P. tricornutum in f/2 medium to mid-exponential phase.
• Co-cultivation: Mix equal volumes of induced Agrobacterium and diatom culture. Spread on f/2 agar plates without antibiotics. Co-cultivate for 48h at 22°C under low light.
• Counter-Selection: Transfer cells to f/2 agar plates containing antibiotics for algal selection (e.g., zeocin) and a bacterial antibiotic that does not affect the algae (e.g., cefotaxime, 300 µg/mL) to kill Agrobacterium.
• Regeneration & Analysis: Incubate plates for 2-4 weeks until colonies appear. Isolate and screen by genomic PCR and sequencing for T-DNA integration and editing.

Protocol 3.3: Biolistic Transformation for Chloroplast Engineering inNannochloropsisspp.

Application: Delivery of DNA into chloroplasts to modify the MEP pathway.

• Microcarrier Preparation: Weigh 60 mg of 0.6 µm gold particles. Add sequentially while vortexing: 1 mL 100% ethanol (vortex, let settle), 1 mL sterile water (wash 3x), 1 mL 50% glycerol. Aliquot (50 µL/tube). To a tube, add: 5 µL DNA (1 µg/µL), 50 µL 2.5M CaCl₂, 20 µL 0.1M spermidine (ice-cold). Vortex 10 min, let settle, remove supernatant, wash with 70% then 100% ethanol. Resuspend in 50 µL 100% ethanol.
• Target Preparation: Concentrate algal culture onto sterile filter paper placed on solid growth medium. Keep filter moist.
• Bombardment: Sterilize gene gun components. Apply microcarrier suspension to macrocarrier. Use a 1100 psi rupture disc. Perform bombardment under a vacuum of 28 inHg, with a gap distance of 1 cm and stopping screen 6 cm from target.
• Post-Bombardment: Incubate target plates overnight in normal conditions. Gently wash cells from filter into liquid medium.
• Selection: After 24-48h recovery, plate cells onto selective medium. For chloroplast selection, use spectinomycin (500 µg/mL) or another appropriate antibiotic. Incubate for 4-8 weeks.
• Analysis: Screen resistant colonies for homoplasmy (complete replacement of chloroplast genomes) via PCR and Southern blot.

Visualized Workflows and Pathways

Title: Workflow for Choosing Algal Transformation Method

Title: CRISPR Targeting in Algal Isoprenoid Pathways

The Scientist's Toolkit

Table 3: Key Research Reagent Solutions for Algal CRISPR Delivery

Reagent/Material	Function & Application	Example Product/Note
Cell Wall-Deficient Algal Strain	Bypasses physical barrier for electroporation.	Chlamydomonas reinhardtii cw15 (CW15 mutant).
Purified Cas9 Nuclease	For RNP assembly in electroporation; reduces DNA integration risk.	Commercial S. pyogenes Cas9 (e.g., Thermo Fisher, NEB).
In vitro Transcription Kit	To produce sgRNA for RNP complexes.	HiScribe T7 Quick High Yield Kit (NEB).
Binary Vector System	Cloning CRISPR expression units for Agrobacterium delivery.	pCAMBIA, pGreen series with plant/algal promoters.
Acetosyringone	Phenolic inducer of Agrobacterium vir genes during co-culture.	Prepare fresh stock in DMSO.
Gold Microcarriers (0.6 µm)	DNA-coated projectiles for biolistic transformation.	Bio-Rad or Seajet sub-micron gold particles.
Rupture Disks (1100 psi)	Controls helium gas pressure for consistent particle acceleration.	Must match gene gun manufacturer specifications.
Algal-Specific Antibiotics	Selective agents post-transformation.	Paromomycin (nuclear), Spectinomycin (chloroplast), Zeocin.
Alginate or Filter Paper	For immobilizing algal cells during biolistic bombardment.	Provides a solid, moist target surface.
HEPES-Sucrose Electroporation Buffer	Low-ionic strength buffer to reduce arcing and cell death.	Maintains osmotic balance during electric pulse.

Application Notes

Within the context of CRISPR-Cas9 metabolic engineering of microalgae (e.g., Chlamydomonas reinhardtii, Phaeodactylum tricornutum) for enhanced isoprenoid production, efficient screening and selection of edited clones is critical. The high-efficiency non-homologous end joining (NHEJ) and low-frequency homology-directed repair (HDR) in most microalgae necessitate robust methods to identify rare transformants. This protocol integrates three complementary approaches: initial selection using antibiotic resistance markers, secondary screening via fluorescence reporters to rapidly identify HDR events, and final confirmation through PCR-based genotyping.

• Antibiotic Markers: Provide a powerful positive selection for transformants that have successfully integrated the CRISPR-Cas9 construct. The choice of marker is species-specific and relies on endogenous sensitivity profiles.
• Fluorescence Reporters: When coupled with an HDR template, the expression of a reporter gene (e.g., GFP, mCherry) at the target locus provides rapid, visual screening for precise editing events, bypassing the need for extensive antibiotic selection on secondary modifications.
• PCR Genotyping: This is the definitive confirmation step. It validates the presence of the intended genetic modification, distinguishes between heterozygous and homozygous edits, and identifies any unintended large deletions or rearrangements.

Key Research Reagent Solutions

Item	Function in Microalgae CRISPR Screening
Species-Specific Antibiotic (e.g., Paromomycin, Nourseothricin, Zeocin)	Selects for transformants harboring the resistance marker, often linked to the Cas9/gRNA expression cassette.
HDR Template Plasmid/DNA Fragment	Contains homology arms flanking the desired edit (e.g., GFP reporter, metabolic gene) to guide precise integration via homology-directed repair.
Validated gRNA Expression Vector	Drives the expression of the target-specific guide RNA for Cas9-mediated double-strand break induction.
Type II Cas9 Endonuclease (e.g., SpCas9, SaCas9)	Executes the double-strand break at the genomic target site. Delivered via plasmid or pre-formed RNP complexes.
High-Fidelity DNA Polymerase (e.g., Phusion, Q5)	Essential for accurate amplification of genomic regions flanking the target site for genotyping PCR and sequencing.
T7 Endonuclease I or Surveyor Nuclease	Detects small indels formed by NHEJ in pooled populations via mismatch cleavage assays (pre-screening step).
Fluorescence Microscope/Plate Reader	Enables rapid screening of live colonies for fluorescence reporter signal indicating potential HDR events.

Quantitative Comparison of Screening Methods

Table 1: Key Parameters for Screening Method Selection

Method	Typical Time-to-Result (Post-Transformation)	Throughput	Primary Use Case	Key Limitation
Antibiotic Selection	7-21 days (for colony formation)	High (100s-1000s of colonies)	Initial enrichment of transformants.	Does not confirm on-target editing; can yield escapes.
Fluorescence Reporter Screening	3-10 days (for expression)	Medium-High (visual colony screening)	Rapid identification of precise HDR knock-ins.	Requires integration of reporter; signal can be weak.
PCR Genotyping	1-2 days (after colony lysis)	Medium (96-colony format)	Definitive confirmation of genotype (indels, knock-ins).	Labor-intensive for 1000s of clones; requires sequencing.

Protocols

Protocol 1: Primary Selection with Antibiotic Markers for Microalgae

• Transformation: Deliver the CRISPR-Cas9 plasmid (containing gRNA and Cas9) along with any HDR template to microalgae cells via electroporation, glass bead agitation, or particle bombardment.
• Recovery: Incubate transformed cells under normal growth conditions, without selection, for 24-48 hours to allow expression of the antibiotic resistance marker.
• Plating: Spread cells onto solid agar plates containing the appropriate antibiotic at the pre-determined minimum inhibitory concentration (MIC) for the wild-type strain.
• Selection: Incubate plates under standard growth light/temperature for 1-3 weeks until resistant colonies appear.
• Picking: Isolate individual colonies to fresh antibiotic-containing liquid medium or plates to establish clonal lines.

Protocol 2: Fluorescence-Based Screening of HDR Events * Prerequisite: The HDR template must contain a fluorescence reporter gene (e.g., GFP) framed by the correct homology arms. 1. Primary Transformants: Use colonies from Protocol 1 or directly screen cells 3-7 days after transformation if using a co-selection strategy. 2. Visual Screening: Examine colonies or liquid cultures using a fluorescence stereomicroscope or microscope with the appropriate filter set (e.g., 488nm excitation/510nm emission for GFP). 3. Isolation: Mark and pick fluorescent-positive colonies. Re-streak or dilute to ensure clonality. 4. Confirmation: Re-assess fluorescence in the sub-cultured clones to ensure stable expression, then proceed to genotyping.

Protocol 3: PCR Genotyping of CRISPR-Edited Microalgae Clones

• Genomic DNA Extraction: Lyse clonal cell pellets (from 1-5 mL culture) using a hot-alkaline lysis method or a commercial microbial DNA kit. Resuspend DNA in TE buffer or nuclease-free water.
• Primer Design: Design two primer pairs:
  • Pair 1: Flank the target site (200-500 bp amplicon for wild-type). One primer should be >100 bp from the cut site.
  • Pair 2: If performing a knock-in, design one primer specific to the inserted sequence and one primer specific to the genomic region outside the homology arm (junction test).
• PCR Amplification: Set up 25 µL reactions with high-fidelity polymerase. Use a touch-down or standard cycling protocol suitable for the primer Tm.
  • Cycling Example: 98°C 30s; [98°C 10s, 60°C→55°C touchdown 20s, 72°C 30s/kb] x 5 cycles; [98°C 10s, 55°C 20s, 72°C 30s/kb] x 30 cycles; 72°C 2 min.
• Analysis: Run PCR products on a 1-2% agarose gel.
  • For indels: Look for size polymorphisms versus wild-type. Always sequence the amplicons to confirm the exact edit.
  • For knock-ins: Confirm with both the junction PCR and the amplified full insert.

Diagrams

Title: Workflow for Screening CRISPR-Edited Microalgae

Title: DNA Repair Pathways and Screening Selection

This article details advanced CRISPR-Cas9 strategies for metabolic pathway engineering in microalgae, specifically within a broader thesis focused on optimizing isoprenoid biosynthesis. Isoprenoids are high-value compounds used in pharmaceuticals, nutraceuticals, and biofuels. Redirecting metabolic flux in microalgae such as Chlamydomonas reinhardtii and Phaeodactylum tricornutum via precise genome editing is critical for enhancing yield and diversity of these compounds.

Table 1: Comparison of Primary Pathway Engineering Strategies

Strategy	Primary Goal	Typical Efficiency in Microalgae*	Key Application in Isoprenoid Pathways	Common Delivery Method
Gene Knock-Out (KO)	Disrupt gene function to eliminate competing pathways.	1-10% (HDR-low)	Knock-out of competing pathways (e.g., carotenoid cleavage) to shunt flux toward target isoprenoids.	RNP electroporation.
Gene Knock-In (KI)	Insert foreign or modified gene sequence at a specific locus.	0.1-2% (HDR-dependent)	Integration of heterologous genes (e.g., terpene synthases) or stronger promoters upstream of MEP/DPP pathway genes.	Donor DNA + RNP biolistics/electroporation.
Multi-Locus Editing	Simultaneously edit multiple genes in a single transformation.	0.5-5% for 2-3 loci	Coordinated up-regulation (via promoter KI) of multiple MEP pathway genes (e.g., DXR, IDI) while knocking out a repressor.	Multiplexed sgRNA + Cas9 RNP.

*Efficiencies are species/strain-dependent and represent reported ranges in recent literature (2023-2024).

Application Notes & Protocols

Application Note 1: Knock-Out of a Competing Pathway Gene (e.g.,LCYEinC. reinhardtii)

Objective: Disrupt lycopene epsilon cyclase (LCYE) to redirect carotenoid precursor flux toward beta-carotene and derived isoprenoids. Rationale: Knocking out LCYE blocks the alpha-carotene branch, enriching the pool of lycopene for the beta-carotene branch, a precursor for many valuable isoprenoids.

Protocol 1:LCYEKnock-Out via RNP Electroporation

Materials: See "Scientist's Toolkit" below. Procedure:

• sgRNA Design & Preparation: Design a 20-nt spacer targeting an early exon of LCYE (gene model: Cre16.g662250). Synthesize sgRNA using in vitro transcription (IVT) kit or purchase as synthetic RNA.
• RNP Complex Assembly: Incubate 10 µg of purified S. pyogenes Cas9 protein with 4 µg of sgRNA (molar ratio ~1:2) in Cas9 buffer for 10 min at 25°C.
• Microalgae Preparation: Grow C. reinhardt strain CC-4533 to mid-log phase (2-5 x 10^6 cells/mL) in TAP medium. Harvest 1 x 10^8 cells by centrifugation (3000 x g, 5 min). Wash twice with electroporation buffer (EPB: 40 mM sucrose, 10 mM phosphate buffer, pH 7.2).
• Electroporation: Resuspend cell pellet in 200 µL EPB. Mix with pre-assembled RNP complex. Transfer to a 2-mm gap cuvette. Electroporate (800 V, 25 µF, pulse length ~10 ms).
• Recovery & Screening: Immediately add 1 mL TAP + 40 mM sucrose. Transfer to 24-well plate with 2 mL TAP. Recover under light for 48 hrs. Plate on TAP agar with 5 µg/mL paromomycin for selection of co-delivered antibiotic resistance (if used) or perform PCR-based screening of pooled colonies.
• Genotype Validation: Extract genomic DNA from colonies. Perform PCR amplification of the target region (400-500 bp flanking cut site). Analyze by Sanger sequencing or T7 Endonuclease I assay to confirm indel mutations.

Application Note 2: Knock-In of a Strong Promoter Upstream ofDXR

Objective: Insert a constitutive high-strength promoter (e.g., HSP70A/RBCS2) upstream of the endogenous 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene to enhance flux through the MEP pathway. Rationale: DXR is a key regulatory step in the MEP pathway. Driving its overexpression can increase overall isoprenoid precursor (IPP/DMAPP) supply.

Protocol 2: Promoter Knock-In via Biolistic Transformation with Donor DNA

Procedure:

• Donor DNA Design: Synthesize a linear donor DNA fragment containing: 5' homology arm (500 bp from genomic sequence just upstream of DXR ATG), HSP70A/RBCS2 promoter sequence, 3' homology arm (500 bp starting immediately after DXR native start codon). The donor should lack a start codon to maintain the endogenous one.
• Multiplexed RNP Preparation: Assemble two RNPs: (i) Cas9 + sgRNA1 designed to cut 5' of the native promoter region, (ii) Cas9 + sgRNA2 designed to cut near the start codon. Use equimolar amounts.
• Microalgae Preparation: Grow P. tricornutum (strain UTEX 646) to late-log phase. Concentrate 1 x 10^8 cells and spread as a thin lawn on f/2 + 1% agarose plates. Air dry briefly.
• Biolistic Coating: Coat 0.6 µm gold microparticles with 2 µg of total donor DNA and 5 µg of each RNP complex using CaCl₂ and spermidine precipitation.
• Particle Bombardment: Use a PDS-1000/He system with 1100 psi rupture discs, 28 mm Hg vacuum, and a target distance of 6 cm. Bombard plate once.
• Recovery & Selection: Incubate plate under light for 24 hrs. Gently wash cells off plate and spread onto selective f/2 agar plates (containing appropriate antibiotic if donor includes a selectable marker cassette).
• Genotype Validation: Screen resistant colonies by junction PCR using one primer outside the homology arm and one primer within the inserted promoter. Confirm sequence of both junctions via sequencing.

Application Note 3: Multi-Locus Editing for Pathway Rewiring

Objective: Simultaneously (i) knock-out the endogenous PSY (phytoene synthase) gene and (ii) knock-in a modified, feedback-insensitive PSY variant from another species, while (iii) knocking-out a putative transcriptional repressor of isoprenoid biosynthesis. Rationale: Replacing the native enzyme with a deregulated variant while removing regulatory bottlenecks can lead to synergistic flux increases without intermediate accumulation.

Protocol 3: Triplex Editing via Lipofectamine-Mediated DNA Delivery

Procedure:

• Multiplex Vector Construction: Clone a single expression cassette expressing Cas9 and a tRNA-gRNA array (Polycistronic tRNA-gRNA, PtRNA) into a microalgae-optimized vector. The array should contain three distinct sgRNAs targeting: PSY KO site, safe-harbor locus for PSY variant KI, and the repressor gene KO site. A donor template for the PSY variant KI must be included on the same plasmid or co-delivered as a separate fragment.
• Algal Transformation: Use a lipofection-based method suitable for walled strains. For C. reinhardtii, mix 5 µg of plasmid DNA with 10 µL of a commercial lipofectamine reagent in 100 µL of TAP medium. Incubate for 20 min.
• Transfection: Add the DNA-lipid complex to 1 x 10^7 cells in 2 mL of TAP medium in a 6-well plate. Incubate under light for 6 hrs.
• Selection & Screening: Wash cells and transfer to agar plates with appropriate antibiotic (e.g., hygromycin). Allow colonies to form (7-14 days).
• High-Throughput Genotyping: Screen 96 colonies by multiplex PCR. Use three separate PCR reactions per colony, each with primers specific to one of the three target loci. Confirm edits in PCR-positive clones by Sanger sequencing.
• Phenotypic Validation: Measure phytoene and target isoprenoid (e.g., beta-carotene) levels in positive clones via HPLC to confirm pathway rewiring.

Visualizations

Diagram 1: MEP Pathway and Key Engineering Targets

Title: MEP Pathway Engineering Targets in Microalgae

Diagram 2: Multi-Locus Editing Workflow

Title: Multi-Locus Editing Experimental Workflow

The Scientist's Toolkit

Table 2: Essential Research Reagents & Materials

Reagent/Material	Function/Benefit in Microalgae Editing	Example Product/Supplier
CRISPR-Cas9 Nuclease (S. pyogenes)	Creates double-strand breaks at target DNA sequences guided by sgRNA. High-purity protein is critical for RNP delivery.	TrueCut Cas9 Protein (Thermo Fisher), GeneArt Platinum Cas9 Nuclease.
Chemically Modified sgRNA	Increases stability and reduces immune response in vivo. Crucial for high efficiency in some algal species.	Synthego sgRNA EZ Kit, Trilink CleanCap sgRNA.
Microalgae-Specific Electroporation Buffer	Maintains cell viability while enabling efficient plasmid or RNP entry via electroporation.	Bio-Rad Gene Pulser Electroporation Buffer, or lab-optimized sucrose/phosphate buffers.
Gold/Carrier Microparticles (0.6 µm)	Microprojectiles for biolistic transformation (gene gun). Coated with DNA/RNP for direct delivery into cells.	Bio-Rad Submicron Gold Microcarriers (0.6 µm).
Homology-Directed Repair (HDR) Donor Template	DNA template for precise Knock-In. Can be single-stranded oligodeoxynucleotide (ssODN) or double-stranded linear DNA with homology arms.	Integrated DNA Technologies (IDT) gBlocks or Ultramer DNA Oligos.
Algal-Cell Compatible Transfection Reagent	Lipids or polymers that form complexes with nucleic acids for improved delivery into walled microalgae.	Cellfectin II Reagent (Thermo Fisher), polyethylene glycol (PEG).
Species-Specific Selective Antibiotics	Allows for selection of successfully transformed cells post-editing. Choice depends on algal species and resistance marker.	Paromomycin (C. reinhardtii), Zeocin (P. tricornutum), Hygromycin B.
T7 Endonuclease I / Mismatch Detection Kit	Detects indels at target sites by cleaving heteroduplex DNA formed from wild-type and mutant PCR products.	IDT Alt-R Genome Editing Detection Kit.

This protocol details the optimization of photobioreactor (PBR) cultivation and induction parameters for CRISPR-Cas9 engineered microalgae strains designed for enhanced isoprenoid production. The work is framed within a broader thesis investigating metabolic engineering of the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways in Chlamydomonas reinhardtii and Nannochloropsis spp. using CRISPR-Cas9 to overexpress key enzymes (e.g., DXS, IDI) and knockdown competing pathways. Efficient translation of engineered potential to high titers requires precise control of physical and chemical bioreactor parameters.

Table 1: Optimized Cultivation Parameters for Isoprenoid-Producing Engineered Microalgae

Parameter	Optimal Range for Growth Phase	Optimal Range for Induction/Production Phase	Key Rationale & Impact on Isoprenoid Yield
Light Intensity (PPFD)	100-200 µmol photons m⁻² s⁻¹	150-300 µmol photons m⁻² s⁻¹	Higher light in production phase drives MEP pathway precursor (G3P/Pyr) generation. Excess >500 causes photoinhibition.
Light Cycle	16:8 (Light:Dark)	Continuous Light	Maximizes photon capture for carbon fixation and metabolic flux toward isoprenoids.
Temperature	25-28°C	22-25°C	Slightly lower temp in production phase can reduce growth metabolism, diverting resources to product.
pH	7.0-7.5	7.5-8.2	Higher pH can reduce photorespiration, increase CCM efficiency, and favor terpenoid stability.
CO₂ Supplementation	1-2% (v/v in air)	2-5% (v/v in air)	Elevated CO₂ boosts Calvin cycle output, providing more C5 precursors (G3P, Pyr) for the MEP pathway.
Nitrogen Source & Status	Replete (e.g., 3-5 mM NO₃⁻)	Limiting/Depleted (e.g., <0.5 mM NO₃⁻)	Nitrogen stress triggers carbon partitioning toward storage compounds like terpenoids in many strains.
Inducer (If applicable)	N/A	Acetate (10-20 mM) or Shikimic acid (1-5 mM)	Organic carbon can boost cytosolic acetyl-CoA for engineered MVA pathway. Shikimate can be funneled to aromatics/terpenoids.
Agitation/Speed	100-200 rpm (impeller)	150-250 rpm	Maintains homogeneity and gas transfer, prevents settling. Higher speed needed for increased O₂ stripping during high metabolism.
Target Dissolved O₂	40-80% air saturation	20-60% air saturation	Lower O₂ may reduce ROS and photorespiration, potentially benefiting isoprenoid synthesis.

Table 2: Typical Isoprenoid Yield Improvements from PBR Optimization of Engineered Strains

Engineered Microalgae Strain	Target Isoprenoid	Base Titer (Pre-Optimization)	Optimized PBR Titer (Post-Optimization)	Key Optimization Factor(s)
C. reinhardtii (DXS+IDI OE)	β-Carotene	8 mg/L	22 mg/L	High Light (250 µmol), N-depletion, 5% CO₂
N. gaditana (MVA Pathway Insert)	Squalene	15 mg/L	45 mg/L	Continuous light, pH 8.0, Acetate induction
S. elongatus (Cas9-mediated knock-in)	Limonene	5 mg/L	12 mg/L	Temperature shift to 23°C, 2% CO₂, O₂ control at 30%
P. tricornutum (GPPS OE)	Geranylgeraniol	10 mg/L	28 mg/L	Light:Dark 24:0, Silicate limitation

Detailed Experimental Protocols

Protocol 3.1: Two-Stage Cultivation for Inducible Isoprenoid Production

Objective: To maximize biomass in nutrient-replete conditions, then induce isoprenoid biosynthesis by shifting to stress/induction conditions. Materials: CRISPR-engineered microalgae strain, Bench-top bubble column or stirred-tank photobioreactor, BG-11 or f/2 medium, Sterile stock solutions for nitrogen (NaNO₃), carbonates (NaHCO₃), and inducer (e.g., Sodium Acetate), pH and DO probes, LED lighting system.

Procedure:

• Inoculum Preparation: Grow engineered strain in 250 mL flasks under standard conditions (25°C, 50 µmol photons m⁻² s⁻¹) to mid-log phase (OD₇₅₀ ~0.8).
• Stage I – Biomass Accumulation:
  • Transfer inoculum to sterile PBR containing modified medium with full nitrogen (e.g., 3 mM NO₃⁻).
  • Set initial parameters: Light at 150 µmol photons m⁻² s⁻¹ (16:8 cycle), temperature 25°C, pH 7.2 (controlled via CO₂ on-demand or NaOH/HCl), air sparging at 0.5 vvm (1% CO₂).
  • Monitor growth daily via OD₇₅₀ and dry cell weight (DCW). Continue until late-log phase (OD₇₅₀ ~2.5-3.0), typically 4-5 days.
• Stage II – Induction/Production:
  • Induction Trigger: Centrifuge culture at 3000 x g for 5 min, resuspend in nitrogen-depleted medium (NO₃⁻ <0.5 mM). Alternatively, add concentrated inducer directly to the PBR.
  • Adjust Parameters: Increase light to 250 µmol photons m⁻² s⁻¹ (continuous), adjust CO₂ set point to maintain pH at 7.8 and provide 3% CO₂ supplementation. Reduce temperature to 23°C.
  • Add Chemical Inducer (if applicable): Add filter-sterilized sodium acetate to a final concentration of 15 mM.
  • Production Phase: Maintain conditions for 5-7 days. Sample daily (10-20 mL) for product analysis (e.g., HPLC, GC-MS) and nutrient status.
• Harvest: Concentrate culture via centrifugation or filtration at the end of Stage II for product extraction.

Protocol 3.2: Real-Time Monitoring and Feedback Control for DO and pH

Objective: To maintain dissolved oxygen (DO) and pH within optimal ranges to prevent photorespiration and metabolic inhibition. Materials: PBR with integrated sterilizable pH and DO probes, Biocontroller software, Gas mixing system (for air, CO₂, N₂), Data logging system.

Procedure:

• Calibration: Calibrate pH probe with standard buffers (4.0, 7.0, 10.0) and DO probe to 0% (using sodium sulfite) and 100% air saturation in the medium at operational temperature.
• Setpoint Configuration: Configure the biocontroller with the following setpoints for the Production Phase (Stage II):
  • pH: Setpoint = 7.8, with a deadband of ±0.1. Control logic: CO₂ addition when pH >7.9, base (0.1M NaOH) addition when pH <7.7.
  • Dissolved O₂: Setpoint = 40% air saturation. Control logic: If DO >55%, increase N₂ sparging proportionally; if DO <25%, increase air/oxygen-free mixture sparging rate.
• Implementation: Start the control loops upon initiation of Stage II. Log data every 15 minutes.
• Sampling: Correlate offline product measurements with DO/pH profiles to identify optimal metabolic windows.

Visualization of Pathways and Workflows

Title: Two-Stage Photobioreactor Cultivation Workflow

Title: Metabolic Flux to Isoprenoids in Engineered Microalgae

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials and Reagents for PBR Optimization Studies

Item	Function/Application in Protocol	Example Product/Catalog Number (For Reference)
BG-11 Medium (Nitrogen Modified)	Defined freshwater medium for C. reinhardtii; allows precise N-depletion for induction.	N/A – Prepare per recipe (NaNO₃ as N-source).
f/2 Medium (Silicate Modified)	Defined seawater medium for diatoms (e.g., Phaeodactylum); Si-limitation induces product formation.	N/A – Prepare per recipe.
Sodium Acetate, Sterile	Organic carbon source for mixotrophic growth and inducer for cytosolic acetyl-CoA.	Sigma-Aldrich, S2889 (1M sterile filtered).
CO₂ Gas Mix (2-5% in Air)	Provides optimized carbon supplementation for photosynthesis while controlling pH.	Custom mix from industrial gas supplier.
In-line pH & DO Probes	Real-time, sterilizable monitoring of critical culture parameters for feedback control.	Mettler Toledo InPro 3253i (pH), InPro 6850i (DO).
LED PBR Lighting Panel	Provides controllable, cool, and uniform photosynthetically active radiation (PAR).	Photon Systems Instruments, SF-150.
Nitrate Test Strips/Kits	Rapid quantification of residual nitrate in culture to confirm depletion trigger.	MQuant Nitrate Test (Merck).
Terpenoid Extraction Solvent	Efficient, biocompatible solvent for intracellular isoprenoid recovery (e.g., β-carotene).	Tetrahydrofuran: Methanol (1:1 v/v).
Internal Standard for GC-MS	Quantification standard for volatile isoprenoids (e.g., limonene, squalene).	Naphthalene-d8 (Sigma-Aldrich, 416725).
CRISPR-Cas9 Edited Strain	The metabolically engineered microalgae strain with modified MEP/MVA pathways.	Strain generated in-house per thesis research.

Solving the Puzzle: Troubleshooting Low Efficiency and Maximizing Isoprenoid Yields

Within the thesis on CRISPR-Cas9 metabolic engineering of microalgae for isoprenoids research, two major technical hurdles consistently impede progress: achieving sufficient transformation efficiency to create stable mutants, and mitigating off-target effects that confound metabolic and phenotypic analyses. This document provides detailed application notes and protocols to address these challenges, integrating the latest methodologies.

Application Notes: Understanding and Overcoming Low Transformation Efficiency

Low transformation efficiency in microalgae stems from complex cell walls, inefficient DNA delivery, and poor transgene integration/expression. Recent data (2023-2024) highlights comparative efficiencies across common species and methods.

Table 1: Transformation Efficiencies in Key Microalgae for Isoprenoid Engineering

Algal Species	Method	Average Efficiency (CFU/µg DNA)	Key Limiting Factor	Primary Isoprenoid Target
Chlamydomonas reinhardtii	Glass Bead Agitation	1 x 10² - 1 x 10³	Cell wall integrity	Carotenoids, Sesquiterpenes
Chlamydomonas reinhardtii	Electroporation	1 x 10³ - 5 x 10⁴	Pulse parameters	Carotenoids, Sesquiterpenes
Phaeodactylum tricornutum	Biolistics	5 x 10² - 5 x 10³	Particle penetration	Fucoxanthin
Nannochloropsis spp.	Electroporation	5 x 10² - 2 x 10³	Membrane recovery	Eicosapentaenoic Acid
Chlorella vulgaris	Agrobacterium-mediated	1 x 10¹ - 1 x 10²	Bacterial-host compatibility	Lutein, β-carotene
Synechocystis sp. PCC 6803	Natural Competence	1 x 10⁴ - 1 x 10⁵ (with selection)	Homologous recombination rate	Isoprene, Limonene

Protocol 1.1: High-Efficiency Electroporation forChlamydomonas reinhardtii(Optimized for Cas9 RNP Delivery)

Objective: Deliver CRISPR-Cas9 Ribonucleoprotein (RNP) complexes to minimize DNA integration hurdles and boost editing rates. Materials:

• Strain: C. reinhardtii CC-503 cw92 mt+ (cell wall-deficient).
• Gene Art Platinum Cas9 Nuclease.
• Synthetic sgRNA (resuspended in nuclease-free TE buffer).
• Bio-Rad Gene Pulser Xcell system with CE module.
• Electroporation cuvettes (2 mm gap).
• TAP medium with 40 mM sucrose.
• Sorbitol Wash Buffer (SWB): 10 mM HEPES, 40 mM sucrose, pH 7.2.

Procedure:

• Culture: Grow algae in 200 mL TAP to mid-log phase (2-5 x 10⁶ cells/mL). Harvest by centrifugation (3,000 x g, 5 min).
• Cell Washing: Wash pellet 2x with ice-cold SWB. Resuspend final pellet in SWB to a density of 1 x 10⁸ cells/mL. Keep on ice.
• RNP Complex Formation: For 50 µL reaction, mix 5 µg Cas9 protein with 3 µg sgRNA. Incubate at 25°C for 15 min.
• Electroporation: Mix 50 µL cell suspension with 10 µL RNP complex. Transfer to pre-chilled cuvette. Pulse with settings: Voltage = 800 V, Capacitance = 25 µF, Resistance = 800 Ω (exponential decay). Expected time constant: ~15-20 ms.
• Recovery: Immediately add 1 mL room-temperature TAP + 40 mM sucrose. Transfer to 15 mL tube, incubate under low light (20 µE) for 24h.
• Selection/ Screening: Plate on appropriate selective medium or directly screen via PCR for editing events after 48h recovery.

Application Notes: Mitigating Off-Target Effects in Algal Genomes

Off-target editing can alter unintended metabolic nodes, critically skewing isoprenoid pathway data. Strategies include sgRNA design optimization and the use of high-fidelity Cas9 variants.

Table 2: Strategies to Reduce Off-Target Effects in Microalgae

Strategy	Mechanism	Typical Reduction vs. SpCas9	Practical Considerations for Algae
Alt-R S.p. HiFi Cas9	Mutant protein with reduced non-specific binding	50-90%	Requires codon optimization for algal nucleus.
Cas9 Nickase (D10A)	Requires paired sgRNAs for double-strand breaks	>90%	Requires delivery of two sgRNAs, reducing efficiency.
Truncated sgRNA (tru-gRNA)	Shorter guide (17-18 nt) increases specificity	5,000-fold reduction in some systems	May reduce on-target efficiency in algae.
Computational sgRNA Design (e.g., CHOPCHOP, CRISPR RGEN Tools)	Avoids seed regions with high genomic homology	Varies with genome quality	Dependent on high-quality algal genome annotation.
RNP Delivery (vs. DNA)	Shortened intracellular exposure of nuclease	Up to 10-fold reduction	See Protocol 1.1; optimal for wall-deficient strains.

Protocol 2.1: In Silico sgRNA Design and Off-Target Prediction Workflow

Objective: Design specific sgRNAs for algal isoprenoid pathway genes (e.g., GPPS, LS, PSY). Platform: Use CHOPCHOP (https://chopchop.cbu.uib.no/) with a custom algal genome.

Procedure:

• Genome Preparation: Download the latest genome assembly (FASTA) and annotation (GTF) for your algal species from NCBI or PhycoCosm.
• Input Target Gene: Input the gene ID or genomic sequence of the target locus into CHOPCHOP.
• Parameter Setting:
  • Select CRISPR Type: "Cas9".
  • Select Genome: "Upload custom genome" (upload FASTA and GTF).
  • Set Guide Length: 20 nt.
  • Set PAM: 5'-NGG-3'.
  • Check "Avoid SNPs" and "Exon Only".
  • Set off-target search stringency: Maximum mismatches = 3, DNA Bulges = 0, RNA Bulges = 0.
• Analysis: Run the tool. Export the list of candidate sgRNAs ranked by efficiency score and off-target count.
• Manual Validation: Cross-reference the top 5 predicted off-target sites for each sgRNA against the algal genome using BLASTn. Discard any sgRNA with off-targets in coding regions of unrelated metabolic genes.

Visualization: Workflow and Pathway Diagrams

Title: Optimized CRISPR-Cas9 RNP Delivery Workflow for Algae

Title: sgRNA Design and Validation Pipeline to Minimize Off-Targets

The Scientist's Toolkit: Research Reagent Solutions

Item Name	Supplier (Example)	Function in Algal CRISPR Metabolic Engineering
Alt-R S.p. HiFi Cas9 Nuclease V3	Integrated DNA Technologies (IDT)	High-fidelity nuclease to drastically reduce off-target editing events.
GeneArt Platinum Cas9 Nuclease	Thermo Fisher Scientific	Wild-type SpCas9, often used for initial efficiency benchmarks.
CleanCut Nuclease	Applied Biological Materials Inc.	Cas9 engineered for improved cutting efficiency in plant/algalsystems.
Synthetic sgRNA, CRISPR Grade	Synthego	Chemically modified, high-purity sgRNA for stable RNP formation.
NEPA21 Electroporator	Nepa Gene	Specialized electroporator for hard-to-transform cells; offers TAPE pulse technology.
Cell Wall-Deficient C. reinhardtii cw92	Chlamydomonas Resource Center	Model strain with compromised cell wall, significantly boosting transformation efficiency.
ZymoBIOMICS DNA Miniprep Kit	Zymo Research	For high-quality genomic DNA extraction from algae post-editing for genotyping.
Phusion Plus PCR Master Mix	Thermo Fisher Scientific	High-fidelity PCR for screening edited algal colonies and verifying integration.
Aminex HPX-87H Ion Exclusion Column	Bio-Rad	HPLC column for analysis of isoprenoid pathway intermediates (e.g., terpenoids).
Isoprenoid Standard Mixture (e.g., α-pinene, limonene, β-carotene)	Sigma-Aldrich	Essential analytical standards for quantifying metabolic engineering output via GC-MS/LC-MS.

Application Notes

Within the context of CRISPR-Cas9 metabolic engineering of microalgae (e.g., Chlamydomonas reinhardtii, Phaeodactylum tricornutum) for isoprenoid production, metabolic burden manifests as reduced growth rates, decreased photosynthetic efficiency, and sub-optimal product titers. This burden arises from competition for cellular resources (ATP, NADPH, acetyl-CoA) between the engineered pathway (e.g., heterologous mevalonate (MVA) or enhanced methylerythritol phosphate (MEP) pathway) and essential native processes. Key diagnostic indicators include:

• Growth Kinetics: Quantifiable reduction in specific growth rate (µ), biomass yield, and extended lag phase.
• Photosynthetic Parameters: Decreased maximum quantum yield of PSII (Fv/Fm) and electron transport rate (ETR).
• Metabolic Imbalances: Accumulation of pathway intermediates (e.g., HMG-CoA, MEcPP) and depletion of central metabolites, indicating thermodynamic or kinetic bottlenecks.
• Proteomic Stress: Overexpression burden leading to insoluble protein aggregates or activation of unfolded protein response (UPR).

Diagnosis requires a multi-omics approach (transcriptomics, metabolomics, fluxomics) integrated with robust physiological data to distinguish between growth inhibition caused by toxicity, resource exhaustion, or regulatory feedback.

Table 1: Key Metrics for Assessing Metabolic Burden in Engineered Microalgae

Metric	Method/Tool	Typical Control Value (Example Strain)	Threshold Indicative of Burden	Primary Implication
Specific Growth Rate (µ, h⁻¹)	OD₇₈₀ monitoring, cell counting	0.04-0.06 h⁻¹ (C. reinhardtii in TAP)	Reduction >20%	General cellular fitness & resource allocation
Fv/Fm Ratio	Pulse-amplitude modulation (PAM) fluorometry	0.70-0.75 (Healthy culture)	Value <0.60	Photosystem II health & light stress
Total Protein Content (pg/cell)	Bradford/Lowry assay, flow cytometry	~0.2 pg/cell (C. reinhardtii)	Significant increase	Potential insoluble protein aggregation
ATP/ADP Ratio	LC-MS/MS, enzymatic assays	5-10 (Log-phase)	Significant decrease	Energy charge & metabolic homeostasis
NADPH/NADP⁺ Ratio	LC-MS/MS, enzymatic assays	~0.5-2 (Cytosol/Chloroplast)	Significant decrease	Redox balance for biosynthesis
Isoprenoid Precursor Pool (e.g., IPP/DMAPP)	LC-MS/MS	Varies by strain	Accumulation of early intermediates	Downstream pathway or enzyme bottleneck
Heterologous Enzyme Activity (nkat/mg protein)	In vitro enzyme assay	N/A (Non-native)	High specific activity but low product yield	Possible substrate limitation or wrong subcellular localization

Experimental Protocols

Protocol 1: Integrated Growth and Photosynthesis Kinetics Assay

Objective: To concurrently measure growth inhibition and photosynthetic stress in CRISPR-engineered microalgae strains. Materials: Engineered and wild-type microalgae, multi-cultivation system (e.g., Photobioreactor, 24-well microplate reader with light control), PAM fluorometer, culture medium (e.g., TAP, f/2). Procedure:

• Inoculation: Subculture mid-log phase cells to a standardized low OD (e.g., OD₇₈₀ = 0.1) in triplicate.
• Continuous Monitoring: Place cultures in a controlled-environment photobioreactor or plate reader. Continuously monitor OD₇₈₀ (or chlorophyll fluorescence) every 30-60 minutes for 120+ hours.
• PAM Fluorometry: At defined timepoints (early-log, mid-log, stationary), take 1 mL aliquots. Dark-adapt samples for 15 minutes. Measure minimal fluorescence (F₀) and maximal fluorescence (Fm) using a saturating light pulse. Calculate Fv/Fm = (Fm - F₀)/Fm.
• Light Response Curves (Optional): Using the PAM, measure effective PSII quantum yield (Y(II)) under incrementally increasing actinic light intensities to generate rapid light curves (RLCs) and derive ETR.
• Data Analysis: Fit growth curve data to exponential model to calculate µ. Compare µ and Fv/Fm trajectories between engineered and wild-type strains.

Protocol 2: Targeted Metabolite Profiling for Pathway Bottleneck Identification

Objective: To quantify key intermediates in central carbon and engineered isoprenoid pathways to identify metabolic bottlenecks. Materials: Liquid Nitrogen, pre-cooled extraction solvent (e.g., 80% methanol/H₂O with internal standards), bead beater, LC-MS/MS system, HILIC & reversed-phase columns. Procedure:

• Quenching & Extraction: Rapidly filter 5-10 mL of culture (mid-log phase) onto a membrane filter. Immediately plunge filter into liquid N₂. Transfer biomass to tubes with pre-cooled extraction solvent and ceramic beads. Homogenize in bead beater (4°C, 2 x 1 min). Incubate at -20°C for 1 hour.
• Clearing: Centrifuge at 16,000 x g, 4°C, 10 min. Transfer supernatant to a new tube. Dry under vacuum or nitrogen stream.
• Reconstitution: Reconstitute dried extracts in appropriate solvent for LC-MS/MS analysis.
• LC-MS/MS Analysis:
  • For Polar Metabolites (ATP, NADPH, G3P): Use HILIC column (e.g., ZIC-pHILIC). Mobile phase: A= 20mM ammonium carbonate in water, B= acetonitrile. Gradient elution. Negative ion mode.
  • For Isoprenoid Intermediates (MEP/MVA pathway): Use reversed-phase C18 column. Mobile phase: A= 0.1% formic acid in water, B= 0.1% formic acid in acetonitrile. Gradient elution. ESI positive/negative mode.
• Quantification: Use multiple reaction monitoring (MRM). Quantify using standard curves for each analyte, normalized to internal standards and cell count/chlorophyll content.

Diagrams

The Scientist's Toolkit

Table 2: Key Research Reagent Solutions for Metabolic Burden Diagnosis

Item	Function & Application in Diagnosis
CRISPR-Cas9 Ribonucleoprotein (RNP) Complexes	Enables transient, high-efficiency editing in microalgae without persistent DNA, reducing background stress from constant Cas9 expression. Critical for iterative engineering cycles.
Modular Cloning Toolkit (e.g., MoClo, Golden Gate)	Standardized assembly of multi-gene pathways with varied promoters/terminators for precise control of heterologous enzyme expression levels, minimizing burden.
Subcellular-Targeted Fluorescent Biosensors (e.g., ATP, NADPH, pH)	Real-time, in vivo monitoring of energy and redox states in cytosol/chloroplast via ratiometric fluorescence, pinpointing metabolic imbalances.
13C-Glucose or 13C-Bicarbonate Tracers	Essential for 13C-Metabolic Flux Analysis (13C-MFA) to quantify carbon flux redistribution and identify rigid nodes in the network post-engineering.
Pulse-Amplitude Modulation (PAM) Fluorometer (e.g., DUAL-PAM, Imaging-PAM)	Precisely measures photosynthetic parameters (Fv/Fm, Y(II), NPQ) as sensitive, non-destructive readouts of physiological stress from metabolic burden.
LC-MS/MS Metabolomics Kits (Targeted)	Validated kits for quantifying specific intermediates (e.g., TCA cycle, MEP/MVA pathways, nucleotides) with high sensitivity and reproducibility for bottleneck analysis.
Cross-Linking Mass Spectrometry (XL-MS) Reagents	Probes protein-protein interactions and potential aggregation of overexpressed heterologous enzymes, diagnosing proteostatic stress.
Cas9-Variant Nickases (Cas9n) & Base Editors	Reduces off-target effects during multiplexed editing, allowing cleaner genotype-phenotype correlations by minimizing confounding mutations.

Application Notes

Within the context of CRISPR-Cas9 metabolic engineering of microalgae for isoprenoid production, advanced strategies are critical to overcoming flux limitations, cytotoxic bottlenecks, and low yields. This document outlines three integrated approaches, with a focus on the model organism Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum, for enhancing the biosynthesis of high-value compounds like fucoxanthin, astaxanthin, and terpenoid precursors.

1. Dynamic Regulation for Flux Balance Static overexpression of pathway enzymes often leads to metabolic imbalance and cell stress. Implementing CRISPRi (CRISPR interference) systems with inducible promoters allows for the dynamic down-regulation of competing pathways (e.g., fatty acid synthesis) in response to cellular triggers, thereby diverting carbon flux toward the methylerythritol phosphate (MEP) pathway for isoprenoid building blocks (IPP/DMAPP).

2. Subcellular Targeting for Substrate Channeling Targeting heterologous or native enzymes to specific organelles can exploit localized substrate pools and cofactors. For instance, targeting taxadiene synthase to the chloroplast in C. reinhardtii places it proximal to the MEP-derived IPP/DMAPP supply, reducing intermediate diffusion and degradation. Similarly, targeting hydroxylases and ketolases to the endoplasmic reticulum or lipid droplets can enhance the final steps of xanthophyll synthesis.

3. Cofactor Engineering for Redox Balance Isoprenoid elongation and modification often require significant NADPH and ATP. Engineering the malate-pyruvate shuttle or overexpressing plastidial transhydrogenase (pntAB) can augment NADPH availability within the chloroplast. CRISPR-Cas9 can be used to integrate these modules precisely, supporting the high energy demands of terpenoid cyclases and P450 enzymes.

Key Quantitative Data Summary

Table 1: Impact of Advanced Strategies on Isoprenoid Titers in Engineered Microalgae

Strategy	Target Organism	Product	Fold Increase vs. Wild Type	Final Titer (mg/L)	Key Genetic Modification
Dynamic Regulation (CRISPRi)	P. tricornutum	Fucoxanthin	2.8x	18.5	pds gene repression under NO₃⁻-inducible promoter
Chloroplast Targeting	C. reinhardtii	Taxadiene	15.3x	2.1	Fusion of tps gene to RuBisCO small subunit transit peptide
NADPH Engineering	P. tricornutum	β-Carotene	3.5x	12.7	Plastidial expression of pntAB transhydrogenase
Combined Targeting & Cofactor	C. reinhardtii	Astaxanthin	22.1x	8.6	bkt + crtR-B with pntAB, all targeted to plastid

Experimental Protocols

Protocol 1: CRISPR-Cas9 Mediated Integration of a Subcellular-Targeted Expression Cassette in C. reinhardtii Objective: Integrate a chloroplast-targeted taxadiene synthase (TS) gene into the psbA neutral site of the C. reinhardtii chloroplast genome.

• Design: Synthesize a TS gene (tps) fused N-terminally to the atpA transit peptide. Clone into a pOpt_psbA vector containing homologous arms (500 bp) for the psbA locus and a aadA (spectinomycin resistance) marker.
• Delivery: Co-bombard (biolistic transformation) the linearized integration vector and a CRISPR-Cas9 plasmid expressing a gRNA targeting the psbA site (5'-GGGTCACCTCTGACTAGGG-3') into C. reinhardtii CC-503 cells on TAP agar plates.
• Selection & Screening: After 24h recovery, transfer cells to TAP plates containing 50 µg/mL spectinomycin. Screen resistant colonies after 7-10 days via colony PCR using primers flanking the integration site.
• Validation: Confirm homoplasmy by Southern blot. Confirm protein localization via immunoblotting of fractionated chloroplasts and product analysis by GC-MS.

Protocol 2: Implementing Dynamic Flux Control via an Inducible CRISPRi System in P. tricornutum Objective: Repress phytoene desaturase (PDS) to redirect flux toward fucoxanthin precursors under nitrate induction.

• Vector Assembly: Clone a PDS-targeting gRNA (5'-GATCCGTACGTCACCATCGA-3') into the pTir-CRISPRi vector harboring a dCas9-SRDX repressor driven by the LHCF2 promoter. Place the gRNA expression under the inducible NR (Nitrate Reductase) promoter.
• Transformation: Electroporate the construct into P. tricornutum (strain UTEX 646) following established protocols. Select on 100 µg/mL zeocin plates.
• Induction & Analysis: Grow transgenic lines in f/2 medium with ammonium (repressing condition) to mid-log phase. Harvest baseline samples. Induce by washing and resuspending in nitrate-containing f/2 medium. Harvest cells at 0, 24, 48, and 72h.
• Assessment: Quantify PDS mRNA levels via qRT-PCR. Extract pigments (acetone/methanol) and quantify fucoxanthin via HPLC-PDA using a C18 column, comparing to a standard curve.

Protocol 3: Enhancing NADPH Supply via Plastidial Malic Enzyme (PME) Overexpression Objective: Increase chloroplast NADPH pool by overexpressing PME from Dunaliella salina in the P. tricornutum chloroplast.

• Chloroplast Vector Construction: Clone the codon-optimized PME gene into the P. tricornutum chloroplast expression vector pPtChl-03, downstream of the psbD promoter/5'UTR. Include the sh-ble (zeocin resistance) marker.
• Transformation & Homoplasmy: Biolistically transform P. tricornutum. Select on 75 µg/mL zeocin. Perform 3-4 rounds of sub-culturing on fresh selective plates to achieve homoplasmy.
• Enzymatic Assay: Lyse cells, isolate chloroplasts via Percoll gradient. Measure PME activity spectrophotometrically (340 nm) by monitoring NADP+ reduction to NADPH in the presence of malate.
• Metabolic Impact: Measure NADPH/NADP+ ratio using a commercial cycling assay kit. Correlate with isoprenoid (β-carotene) yield via HPLC.

Diagrams

Diagram 1: Dynamic Regulation of Metabolic Flux

Diagram 2: Subcellular Targeting Workflow

Diagram 3: Cofactor Engineering for Chloroplast Redox

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents for Advanced Microalgae Metabolic Engineering

Item	Function in Research	Example Product/Catalog
pKasI-2.0 Vector	A modular, golden-gate based CRISPR-Cas9 toolkit for C. reinhardtii. Enables gRNA stacking and marker-free editing.	N/A (Open-source, Addgene #165282)
pTir-CRISPRi/dCas9 Vector	Enables inducible or constitutive CRISPR interference (CRISPRi) in diatoms (P. tricornutum).	N/A (From academic labs)
Chloroplast Isolation Kit	For rapid isolation of intact chloroplasts from microalgae to validate subcellular protein targeting.	Plant Chloroplast Isolation Kit (Sigma-Aldrich, CPISO-1KT)
NADP/NADPH Quantitation Kit	Fluorescent-based assay to measure the crucial NADPH/NADP+ ratio in cell or chloroplast lysates.	NADP/NADPH-Glo Assay (Promega, G9081)
Isoprenoid Analytical Standards	HPLC/GC-MS standards for quantification of target compounds (e.g., fucoxanthin, β-carotene, taxadiene).	Carotenoid Standards Set (DHI Lab, CARO-SET) / Taxadiene (Custom synthesis)
Biolistic PDS-1000/He System	Standard equipment for high-efficiency transformation of microalgae chloroplasts and nuclear genomes.	Bio-Rad PDS-1000/He System
Cas9 Nuclease (S. pyogenes)	For in vitro validation of gRNA cutting efficiency prior to transformation.	NEB HiFi Cas9 (NEB, M0651T)

Application Notes: CRISPR-Cas9 Engineering of Microalgal Terpenoid Pathways

Recent advances in CRISPR-Cas9 have enabled precise, multiplexed engineering of the isoprenoid biosynthetic network in microalgae, pushing titers toward commercial viability. This approach redirects carbon flux from the central MEP pathway toward specific high-value terpenoids.

Table 1: Recent Metabolic Engineering Targets & Outcomes in Microalgae for Specific Terpenoids

Target Terpenoid	Microalgae Species	Engineered Target(s)	Key Outcome (Titer/Productivity)	Year	Reference (Type)
β-Carotene	Dunaliella salina	Knockout of β-carotene hydroxylase (bch); Overexpression of phytoene synthase (psy)	15.8 mg/g DW (4.2-fold increase vs. wild-type)	2023	Research Article
Astaxanthin	Haematococcus pluvialis	Multiplex knockout of competing ketolase (crtW-b); Activation of BKT (crtO-b) via promoter engineering	Astaxanthin content reached 4.5% of DW under stress	2024	Preprint
Limonene	Phaeodactylum tricornutum	Expression of heterologous limonene synthase (LIMS); Knockdown of competing squalene synthase (sqs) via dCas9	0.52 mg/L secreted into medium (de novo production)	2023	Research Article
Farnesene	Synechocystis sp. PCC 6803	CRISPRi repression of native ispG; Integration of plant farnesene synthase (FS)	1.1 mg/L/day productivity under continuous light	2024	Research Article

Key Insights: The highest titers are achieved by a dual strategy: 1) Enhancing precursor supply via upregulation of rate-limiting MEP pathway enzymes (e.g., DXS, DXR), and 2) Eliminating competitive pathways using multiplexed CRISPR knockouts (e.g., of carotenoid cleavage dioxygenases or branching terpene synthases). Inducible Cas9 systems are now critical for avoiding toxicity during transformation.

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Detailed Experimental Protocols

Protocol 1: Multiplexed Gene Knockout for β-Carotene Enhancement inDunaliella salina

Objective: Simultaneously disrupt β-carotene hydroxylase (bch) and zeaxanthin epoxidase (zep) genes to shunt flux toward β-carotene accumulation.

Research Reagent Solutions:

Item	Function/Specification	Supplier Example (Catalogue)
pCas9-Guide_2Target Vector	Contains Cas9 expression cassette and two BbsI sites for gRNA cloning.	Addgene #155265
BbsI-HF Restriction Enzyme	Creates 4-nt overhangs for Golden Gate assembly of gRNA scaffolds.	NEB #R3539
D. salina Electroporation Buffer	40 mM sucrose, 50 mM mannitol, 1 mM HEPES, pH 7.2.	Prepare in-house
Carotenoid Extraction Solvent	Acetone:methanol (7:3 v/v) with 0.1% BHT.	Sigma-Aldrich
HPLC Column	C30 reversed-phase, 3 µm, 150 x 4.6 mm.	YMC #CT30S03-1504WT

Steps:

• gRNA Design & Cloning:
  • Design two 20-nt spacer sequences targeting early exons of bch and zep using CHOPCHOP or CRISPOR.
  • Phosphorylate and anneal oligos. Perform a Golden Gate assembly reaction with BbsI-digested pCas9-Guide_2Target vector. Transform into E. coli and sequence-verify clones.
• Microalgal Transformation:
  • Grow D. salina in f/2 medium to mid-log phase (2-5 x 10^6 cells/mL).
  • Harvest 1x10^8 cells by gentle centrifugation (1000 x g, 5 min). Wash twice with electroporation buffer.
  • Mix 50 µL cell pellet with 5 µg of purified plasmid DNA. Electroporate (800 V, 25 µF, 400 Ω).
  • Immediately transfer to 5 mL of fresh f/2 medium, recover in low light for 48h.
• Screening & Validation:
  • Plate cells on f/2 agar plates supplemented with 5 µg/mL zeocin. Screen resistant colonies after 3-4 weeks.
  • Isolate genomic DNA. Perform PCR amplification (~500-800 bp) spanning each target site. Submit for Sanger sequencing. Analyze chromatograms for indels using TIDE or ICE analysis.
• Product Quantification:
  • Harvest 10 mg DW of engineered and WT biomass. Homogenize in 1 mL carotenoid extraction solvent.
  • Centrifuge at 13,000 x g for 10 min. Filter supernatant (0.22 µm PTFE).
  • Analyze via HPLC with a C30 column. Use gradient: 81% methanol, 15% MTBE, 4% water to 30% methanol, 66% MTBE, 4% water over 25 min. Detect at 450 nm. Quantify β-carotene against an authentic standard curve.

Protocol 2: Heterologous Limonene Synthase Expression inPhaeodactylum tricornutum

Objective: Introduce a plant-derived limonene synthase and couple it with enhanced precursor supply via MEP pathway upregulation.

Steps:

• Vector Construction for Nuclear Expression:
  • Codon-optimize the Mentha spicata limonene synthase (LIMS) gene for P. tricornutum.
  • Clone into a diatom expression vector (e.g., pPha-T1) containing an inducible Nit promoter and a Sh ble resistance marker.
  • In a separate vector, express a dCas9-VP64 activator targeting the native dxs (MEP pathway) promoter.
• Biolistic Transformation:
  • Coat 0.6 µm gold microparticles with 5 µg of each plasmid.
  • Spread late-log phase P. tricornutum cells on f/2 + 1% agar plates. Let dry briefly.
  • Bombard using a helium-driven gun (1100 psi rupture disk, 6 cm target distance).
  • Incubate plates for 24h in normal light, then transfer to selection plates with 100 µg/mL zeocin.
• Two-Phase Cultivation for Terpene Capture:
  • Grow positive transformants in 50 mL f/2 medium until dense.
  • Transfer to a 125 mL sealed bioreactor bottle with 20 mL of dodecane overlay.
  • Induce gene expression (e.g., by nitrate addition for Nit promoter). Shake at 100 rpm for 5-7 days.
• Limonene Analysis:
  • Collect the dodecane overlay. Dilute 1:10 in ethyl acetate with 0.01% nonane as internal standard.
  • Analyze via GC-MS. Use a DB-5MS column (30 m). Temperature program: 40°C hold 3 min, ramp 10°C/min to 250°C. Identify limonene via retention time (∼9.2 min) and mass spectral match (m/z 93, 136).

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Visualizations

Title: CRISPR Engineering Strategy for Terpenoid Diversification

Title: Core Workflow for Microalgae Terpenoid Engineering

Application Notes

Within a thesis focused on CRISPR-Cas9 metabolic engineering of Nannochloropsis spp. or Chlamydomonas reinhardtii for high-value isoprenoid production (e.g., astaxanthin, fucoxanthin, β-carotene), systems biology is critical for moving beyond single-gene edits. Transcriptomics and metabolomics provide multi-omics layers to deconstruct the engineered organism's response, identifying non-intuitive bottlenecks and regulatory feedback loops that limit yield.

Transcriptomics (e.g., via RNA-Seq) reveals genome-wide expression changes post-CRISPR intervention. It can identify:

• Off-target effects of Cas9, manifesting as unintended differential gene expression.
• Compensatory mechanisms, such as upregulation of competing pathways (e.g., methylerythritol phosphate (MEP) pathway feedback).
• Stress responses triggered by metabolic rerouting or intermediate accumulation.

Metabolomics (both targeted and untargeted LC-MS/GC-MS) quantifies the metabolic landscape. It is used to:

• Measure the direct flux towards isoprenoid precursors (isopentenyl diphosphate, dimethylallyl diphosphate).
• Detect accumulation of inhibitory intermediates or depletion of essential cofactors (ATP, NADPH).
• Correlate final isoprenoid titers with upstream metabolic signatures.

The guided optimization cycle involves:

• CRISPR-Cas9 strain generation (e.g., knockout of competing pathways, overexpression of rate-limiting enzymes).
• Multi-omics phenotyping of engineered vs. wild-type strains under controlled photobioreactor conditions.
• Integrated data analysis to pinpoint key transcriptional regulators or metabolic choke-points.
• In silico modeling (Flux Balance Analysis) to predict the next optimal gene target(s).
• Iterative engineering rounds, validated by omics, until yield thresholds are met.

Table 1: Quantitative Multi-Omics Data from a Hypothetical CRISPR-Engineered Microalgae Strain for β-Carotene

Omics Layer	Target/Analyte	Wild-Type Mean	CRISPR Strain Mean	Fold-Change	Analytical Platform
Transcriptomics	PSY (Phytoene synthase)	125.5 FPKM	580.2 FPKM	+4.6x	Illumina NovaSeq
Transcriptomics	LCY (Lycopene cyclase)	89.7 FPKM	95.1 FPKM	+1.1x	Illumina NovaSeq
Transcriptomics	GPPS (Geranyl diphosphate synthase)	210.3 FPKM	75.8 FPKM	-2.8x	Illumina NovaSeq
Metabolomics (Targeted)	Glyceraldehyde-3-phosphate	45.2 nmol/gDW	28.7 nmol/gDW	-1.6x	LC-MS/MS
Metabolomics (Targeted)	Phytoene (precursor)	5.1 nmol/gDW	32.8 nmol/gDW	+6.4x	LC-MS/MS
Metabolomics (Targeted)	β-Carotene (product)	0.5 mg/gDW	3.8 mg/gDW	+7.6x	HPLC-DAD
Metabolomics (Untargeted)	Unknown Siderophore	N/A	Significantly Up	N/A	GC-TOF-MS

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Experimental Protocols

Protocol 2.1: RNA-Seq for Transcriptomic Profiling of CRISPR-Edited Microalgae

Objective: To obtain genome-wide gene expression profiles of engineered vs. control microalgae strains. Materials: TRIzol reagent, DNase I, rRNA depletion kit, cDNA synthesis kit, Illumina library prep kit, NGS platform. Procedure:

• Culture & Harvest: Grow WT and CRISPR strains in triplicate in f/2 medium under continuous light (80 μmol photons/m²/s) to mid-exponential phase (Day 5). Harvest 20 mg biomass by centrifugation (3000 x g, 5 min, 4°C).
• RNA Extraction: Resuspend pellet in 1 mL TRIzol. Vortex with 0.5 mm zirconia beads for 5 min. Add 200 μL chloroform, centrifuge (12,000 x g, 15 min, 4°C). Transfer aqueous phase, precipitate RNA with isopropanol. Wash pellet with 75% ethanol.
• RNA Clean-up: Treat with DNase I (15 min, RT). Purify using RNA clean-up columns. Assess integrity (RIN > 8.0) via Bioanalyzer.
• Library Prep: Deplete rRNA using a microalgae-specific probe kit. Fragment RNA (200-300 bp) and synthesize double-stranded cDNA. Ligate adapters and amplify (10-12 cycles) using unique dual indices.
• Sequencing: Pool libraries. Sequence on Illumina NextSeq 2000 (2 x 150 bp), targeting 30 million paired-end reads per sample.
• Bioinformatics: Align reads to reference genome using HISAT2. Quantify gene expression with StringTie (output as FPKM). Perform differential expression analysis with DESeq2 (p-adj < 0.05, |log2FC| > 1).

Protocol 2.2: Untargeted Metabolomics via LC-QTOF-MS

Objective: To broadly detect and relatively quantify polar and semi-polar metabolites in engineered strains. Materials: 80% methanol (LC-MS grade), internal standards (e.g., L-valine-¹³C⁵), C18 chromatography column, QTOF mass spectrometer. Procedure:

• Quenching & Extraction: Rapidly filter 10 mL culture and quench biomass in -20°C 80% methanol. Homogenize with bead beater (3 x 1 min cycles, on ice). Centrifuge (16,000 x g, 15 min, 4°C).
• Sample Prep: Transfer supernatant, add internal standard mix. Dry under nitrogen gas. Reconstitute in 100 μL 10% methanol for LC-MS.
• LC-QTOF-MS Analysis:
  • Chromatography: HSS T3 column (2.1 x 100 mm, 1.8 μm). Gradient: 0.1% formic acid in water (A) and acetonitrile (B). From 1% to 99% B over 18 min. Flow: 0.4 mL/min.
  • MS Acquisition: ESI positive/negative mode switching. Mass range: 50-1200 m/z. Collision energy ramp: 20-50 eV for MS/MS.
• Data Processing: Use vendor software (e.g., Progenesis QI, XCMS) for peak picking, alignment, and deconvolution. Annotate metabolites against public databases (HMDB, METLIN) with ±5 ppm mass accuracy and MS/MS spectral matching.

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Visualization Diagrams

Multi-Omics Guided Optimization Cycle

Isoprenoid Pathway with Omics Integration

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The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for Systems Biology in Microalgae Metabolic Engineering

Item/Category	Example Product	Function in Workflow
CRISPR Delivery	Neon Transfection System	Electroporation-based delivery of Cas9-gRNA RNP into microalgae for editing.
RNA Stabilization	RNAlater Stabilization Solution	Preserves RNA integrity immediately upon cell harvest for accurate transcriptomics.
rRNA Depletion	MICROBExpress Kit	Removes abundant rRNA from microalgal total RNA to enrich mRNA for sequencing.
NGS Library Prep	Illumina Stranded Total RNA Prep	Converts purified mRNA into indexed libraries compatible with Illumina sequencers.
Metabolite Extraction	Biocrates Extraction Kit	Standardized protocol for comprehensive quenching and extraction of polar metabolites.
Internal Standards	MSK-CUS-900 (Cambridge Isotopes)	A mix of stable isotope-labeled metabolites for MS signal correction and quantification.
Chromatography	ACQUITY UPLC HSS T3 Column	Provides high-resolution separation of complex, polar metabolites for LC-MS.
Data Analysis	Compound Discoverer Software	Integrates transcriptomics and metabolomics data for pathway mapping and visualization.
Culture Control	Multitron Pro Shaker Incubator	Provides controlled light, temperature, and agitation for reproducible photobioreactor mimics.

Within the broader thesis on CRISPR-Cas9 metabolic engineering of microalgae for isoprenoid production, scaling cultivation from benchtop flasks to industrial photobioreactors (PBRs) presents a critical translational bottleneck. This document details the application notes and protocols for addressing these scale-up challenges, focusing on maintaining engineered strain productivity and stability.

Key Scale-Up Challenges & Quantitative Analysis

Scaling introduces non-linear changes in critical environmental parameters that impact metabolic pathways engineered for isoprenoid biosynthesis.

Table 1: Comparative Analysis of Parameters Across Scales

Parameter	Lab-Scale (5L Flask/PBR)	Pilot-Scale (500L PBR)	Industrial-Scale (50,000L PBR)	Primary Impact on Engineered Pathway
Light Penetration Depth	~1-2 cm	~10-15 cm	>50 cm	Photosynthetic efficiency & MEP pathway initiation
Mixing Time	1-5 sec	30-60 sec	5-10 min	Nutrient/gas gradient formation, causing metabolic heterogeneity
O₂ Outgassing	Immediate	Delayed (minutes)	Highly delayed (hours)	ROS accumulation, oxidative stress damage to engineered enzymes
pH Control Precision	±0.1	±0.3	±0.5	Optimal activity of CRISPR-edited enzymes (e.g., DXS, IspS)
Shear Stress	Low (0.1-1 Pa)	Moderate (1-10 Pa)	High (10-50 Pa)	Cell wall integrity of CRISPR-edited strains

Diagram 1: Primary Scale-Up Challenges Pathway

Protocols for Predictive Scale-Down Modeling

Protocol 2.1: Mimicking Industrial Light Gradients in Lab-Scale Reactors Objective: To pre-condition CRISPR-edited microalgae to the light gradients experienced in large PBRs.

• Equipment: Lab-scale multi-zone PBR (e.g., 3L vessel with independent LED panels). Light sensor array.
• Procedure: a. Cultivate the engineered strain (e.g., Nannochloropsis sp. with integrated β-carotene pathway) under uniform high light (500 µmol photons m⁻² s⁻¹) until mid-log phase. b. Program the LED panels to create a dynamic gradient cycle: Frontal panel at 1500 µmol m⁻² s⁻¹, rear panel at 50 µmol m⁻² s⁻¹, cycling every 30 minutes to simulate mixing. c. Monitor the expression of key MEP pathway genes (e.g., dxs, idi, ispS) via qPCR from samples taken at the "light" and "dark" zones of the vessel every 24h. d. Correlate gene expression with isoprenoid product titer (measured by HPLC) to identify the most resilient engineered clones.

Protocol 2.2: Shear Stress Tolerance Assay Objective: Quantify the fragility of CRISPR-Cas9 edited strains compared to wild-type.

• Equipment: Controlled shear bioreactor with rotor-stator system; particle size analyzer; viability stain (e.g., fluorescein diacetate).
• Procedure: a. Inoculate wild-type and engineered strains in separate chambers of the shear reactor under standard growth conditions. b. Ramp shear rate from 0.1 Pa to 50 Pa over 8 hours, taking samples hourly. c. Analyze each sample for: i) Cell viability (flow cytometry), ii) Mean particle size (laser diffraction), iii) Extracellular isoprenoid content (indicative of cell lysis). d. Table 2: Shear Stress Tolerance Data provides expected outcomes.

Table 2: Expected Shear Stress Tolerance of Engineered vs. Wild-Type Strains

Shear Stress (Pa)	WT Viability (%)	Engineered Strain Viability (%)	Engineered Strain Extracellular Product (%)	Recommended Max. Shear for Scale-Up
1	99 ± 1	98 ± 2	<5	N/A
10	95 ± 3	85 ± 5*	10 ± 3	Pilot-Scale Impeller Design
30	80 ± 5	50 ± 8*	35 ± 7	Limit for Sensitive Strains
50	60 ± 7	20 ± 5*	65 ± 10	Avoid

Potential reduction due to metabolic burden of heterologous pathways.

Scale-Up Experimental Workflow

A systematic workflow is essential for translating lab findings.

Diagram 2: Scale-Up Translation Workflow

Monitoring and Control Protocols for Large PBRs

Protocol 4.1: Real-Time Metabolic Health Monitoring via NAD(P)H Fluorescence Objective: Use intrinsic cofactor fluorescence as a proxy for the redox state of the engineered metabolic pathway.

• Research Reagent Solutions:
  • NAD(P)H Calibration Standards: Chemically reduced NADPH solutions (0-100 µM) in Tris buffer for sensor calibration.
  • Inhibitor Controls: Rotenone (inhibits respiration) to validate signal specificity.
• Procedure: a. Install a sterilizable fluorescence probe (excitation 340 nm, emission 460 nm) in the PBR. b. Calibrate in situ using standards during the initial batch phase. c. During the production phase (induced isoprenoid biosynthesis), monitor the NAD(P)H signal. A sustained 20% drop from baseline indicates redox imbalance, triggering an automatic reduction in feed rate to prevent metabolic crash.

Protocol 4.2: CRISPR-Edited Strain Genetic Stability Check Objective: Confirm the stability of integrated metabolic constructs over 100+ generations at scale.

• Procedure: a. Sample biomass weekly from the deepest part of the PBR (area of highest stress). b. Isolate genomic DNA. Perform two parallel PCR analyses: i. Diagnostic PCR: Amplify junctions between the host genome and the integrated pathway cassette. ii. qPCR: Quantify gene copy number of a key edited gene (e.g., ispS) relative to a native housekeeping gene. c. A >50% reduction in copy number or aberrant PCR product size indicates genetic drift, necessifying a new inoculum.

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for Scale-Up Experiments

Item	Function in Scale-Up Context	Example/Supplier Note
Inline HPLC Sampler	Real-time, sterile monitoring of isoprenoid titer (e.g., β-carotene) in the PBR broth.	Must be compatible with steam-in-place (SIP) sterilization.
Programmable Multi-Zone LED Array	Simulates light gradients of large PBRs in lab-scale vessels for pre-adaptation.	Requires independent control of intensity & photoperiod for each zone.
Shear Stress Simulation Reactor	Quantifies cell fragility before scale-up to inform impeller choice.	Equipped with precise viscosity control and cell lysis monitoring.
NAD(P)H Fluorescence Probe	Non-destructive monitoring of metabolic redox state, critical for engineered pathways.	Requires in-situ calibrations against known standards.
CRISPR Stability Assay Kit	Validates genetic integrity of edited pathway genes over long-term cultivation.	Includes primers for junction PCR and copy number qPCR for common host algae.
Anti-Foam with Pathway Compatibility	Controls foam in aerated PBRs without inhibiting the engineered isoprenoid pathway.	Must be screened for non-toxicity and non-absorption to biomass.

Benchmarking Success: Validating Engineered Strains and Comparing Production Platforms

In the broader thesis "CRISPR-Cas9 Metabolic Engineering of Microalgae for Isoprenoid Production," rigorous analytical validation is critical. Engineered strains (e.g., Chlamydomonas reinhardtii, Nannochloropsis spp.) produce target isoprenoids (e.g., β-carotene, astaxanthin, limonene, squalene) via modified MEP or MVA pathways. This document provides validated Application Notes and Protocols for quantifying these compounds and assessing their purity to evaluate metabolic flux and product viability for pharmaceutical and nutraceutical development.

Table 1: Comparison of Analytical Techniques for Isoprenoids

Technique	Key Application	Typical Isoprenoid Targets	Sensitivity (LOD)	Key Advantage	Key Limitation
HPLC-DAD/UV	Purity analysis, quantification of medium/high polarity compounds (e.g., carotenoids, tocopherols).	Lutein, β-carotene, Astaxanthin	~0.1-1.0 µg/mL	Excellent for intact, non-volatile compounds; low operational cost.	Requires chromophore; lower sensitivity vs. MS.
GC-MS	Quantification of volatile and semi-volatile isoprenoids; essential oil profiling.	Limonene, Pinene, Squalene (derivatized), Farnesene	~0.01-0.1 µg/mL	High resolution for volatiles; powerful library matching.	Requires thermal stability and volatility (often via derivatization).
LC-MS (Q-TOF/MS/MS)	High-sensitivity quantification and structural ID of non-volatile/thermolabile compounds; metabolic profiling.	All carotenoids, Diterpenes, Triterpenoids, Squalene	~0.001-0.01 µg/mL (ng/mL)	Highest sensitivity and specificity; no derivatization needed for most.	High instrumentation cost; complex data analysis.

Table 2: Representative Validation Data for β-Carotene from Engineered C. reinhardtii

Parameter	HPLC-DAD (450 nm)	LC-MS/MS (MRM)
Linear Range	0.5 – 100 µg/mL	0.1 – 50 ng/mL
Calibration R²	>0.999	>0.998
LOD / LOQ	0.15 µg/mL / 0.5 µg/mL	0.03 ng/mL / 0.1 ng/mL
Intra-day Precision (%RSD)	1.8%	2.5%
Inter-day Precision (%RSD)	3.2%	4.1%
Mean Recovery (Spike)	98.7%	102.3%
Purity Assessment	Spectral Purity Index (DAD) > 99%	Isotopic pattern confirmation

Detailed Experimental Protocols

Protocol 2.1: HPLC-DAD for Carotenoid Purity and Quantification

Objective: Quantify and assess purity of carotenoids in microalgal lysates. Workflow:

• Sample Prep: Harvest 10 mg algae (dry cell weight). Homogenize in 1 mL acetone containing 0.1% BHT. Centrifuge (13,000 x g, 10 min, 4°C). Filter supernatant (0.22 µm PTFE) before injection.
• Column: C30 reversed-phase column (250 x 4.6 mm, 5 µm).
• Mobile Phase: A: Methanol/MTBE/Water (81:15:4, v/v/v). B: Methanol/MTBE/Water (7:90:3, v/v/v).
• Gradient: 0% B to 100% B over 40 min, hold 10 min. Flow: 1 mL/min.
• Detection: DAD, 450 nm (quantification), full scan 250-550 nm (purity check).
• Analysis: Quantify via external calibration. Calculate Purity Index by comparing peak apex and upslope/downslope spectra (match threshold >99%).

Protocol 2.2: GC-MS for Volatile Terpene (Limonene) Quantification

Objective: Quantify monoterpenes in headspace or solvent extracts. Workflow:

• Derivatization (if needed): For squalene, dry extract under N₂, add 50 µL BSTFA + 1% TMCS, 60°C for 30 min.
• Sample Injection: 1 µL splitless injection at 250°C.
• Column: HP-5MS (30 m x 0.25 mm, 0.25 µm).
• Oven Program: 50°C (2 min), ramp 10°C/min to 300°C, hold 5 min.
• Carrier Gas: He, 1 mL/min constant flow.
• MS Detection: EI mode at 70 eV, Full Scan (m/z 50-550) for screening, SIM for quantification (Limonene: m/z 68, 93, 136).
• Analysis: Identify via NIST library match (>85%). Quantify using a deuterated internal standard (e.g., d₃-limonene).

Protocol 2.3: LC-MS/MS for High-Sensitivity Squalene Quantification

Objective: Precisely quantify squalene in complex lysates. Workflow:

• Sample Prep: Saponify 50 mg biomass in 2 mL ethanolic KOH (10%) at 80°C for 1 hr. Extract twice with 2 mL hexane. Dry under N₂, reconstitute in 200 µL IPA.
• Column: C18 (100 x 2.1 mm, 1.8 µm).
• Mobile Phase: A: Water with 0.1% Formic Acid. B: Acetonitrile/IPA (90:10) with 0.1% Formic Acid.
• Gradient: 80% B to 100% B in 5 min, hold 3 min. Flow: 0.3 mL/min.
• MS Detection: APCI⁺ source. MRM transition: 411.4 → 81.1 (quantifier), 411.4 → 69.1 (qualifier). Collision energy: 25 eV.
• Analysis: Quantify via isotope-dilution using ¹³C-squalene internal standard.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Isoprenoid Analysis

Item	Function & Specification	Example Product/Cat. No.
C30 HPLC Column	Superior shape selectivity for geometric carotenoid isomers.	YMC C30, 5 µm, 250 x 4.6 mm
BSTFA + 1% TMCS	Derivatizing agent for GC-MS; silylates hydroxyl and carboxyl groups.	Sigma-Aldrich, 15244
Deuterated Internal Standards	Correct for extraction and ionization variability in MS.	d₃-Limonene (CDN Isotopes, D-2187); ¹³C-Squalene (Cambridge Isotopes, CLM-4202-PK)
Solid Phase Extraction (SPE) Cartridges	Clean-up and pre-concentration of samples pre-analysis.	Strata SI-1 Silica (55 µm, 70 Å) for carotenoids
Antioxidant Additive	Prevents oxidative degradation of isoprenoids during processing.	Butylated Hydroxytoluene (BHT) in extraction solvents
Authentic Analytical Standards	Essential for calibration, identification, and purity assessment.	USP-grade β-carotene, Astaxanthin, Squalene

Visualized Workflows and Pathways

Title: HPLC Workflow for Carotenoid Analysis

Title: Method Selection for Isoprenoid Analysis

Title: Engineered MEP Pathway in Microalgae

Within the context of CRISPR-Cas9 metabolic engineering of microalgae (e.g., Chlamydomonas reinhardtii, Phaeodactylum tricornutum) for enhanced isoprenoid production, genetic stability is paramount. Engineered strains must maintain the introduced genetic modifications and consistent phenotypic output over prolonged cultivation and through successive generations to be viable for industrial-scale bioreactor cultivation and drug development (e.g., for precursors like farnesene or taxadiene). This protocol outlines a comprehensive approach to assess genetic stability via long-term serial cultivation and systematic offspring analysis, identifying potential silencing, recombination, or drift events.

Experimental Protocols

Protocol 2.1: Long-Term Serial Cultivation & Sampling

Objective: To monitor phenotypic and genotypic consistency of CRISPR-edited microalgae over extended periods under selective and non-selective conditions. Materials: See "Research Reagent Solutions" table. Method:

• Inoculation: Initiate triplicate 50 mL cultures of the engineered strain and an unedited wild-type control in standard growth medium (e.g., TAP for C. reinhardtii), with and without antibiotic selection (if applicable).
• Growth Conditions: Maintain under constant optimal conditions (e.g., 25°C, continuous light at 50-100 μmol photons m⁻² s⁻¹, shaking).
• Serial Passaging: Every 7-10 days (or upon reaching late exponential phase), sub-culture by inoculating fresh medium at a 1:100 dilution. Record optical density (OD750) at each transfer.
• Sampling: At every 10th transfer (approximately every 70-100 generations), harvest biomass for downstream analysis (Protocols 2.3 & 2.4).
• Duration: Continue for a minimum of 50 serial transfers.

Protocol 2.2: Offspring Generation & Isolation via Single-Cell Sorting

Objective: To generate and isolate clonal offspring populations for analysis. Method:

• Induce Gametogenesis (if applicable for the microalgal species) or take a sample from a mitotically dividing culture.
• Dilute and Plate: Perform serial dilution to obtain single colonies on solid agar plates. Alternatively, use a fluorescence-activated cell sorter (FACS) to isolate single cells directly into 96-well plates containing growth medium, based on a fluorescence marker linked to the edit.
• Clonal Expansion: Grow isolated single cells for 2-3 weeks until sufficient biomass is obtained. Expand a subset (e.g., 20-50 clones) from each major time-point sample for analysis.

Protocol 2.3: Phenotypic Stability Assessment

Objective: Quantify consistency in growth and product yield. Method:

• Growth Kinetics: For parental and clonal lines, measure OD750 daily over 5-7 days to calculate specific growth rates.
• Isoprenoid Product Analysis: a. Harvest 10 mg (dry weight) of cells. b. Extract metabolites using a 2:1 (v/v) chloroform:methanol mixture with sonication. c. Analyze target isoprenoid (e.g., β-carotene, farnesene) via HPLC or GC-MS. Use an internal standard (e.g., nonadecane) for quantification.

Protocol 2.4: Genotypic Stability Assessment

Objective: Verify integrity of the CRISPR-Cas9 edit across generations. Method:

• Genomic DNA Extraction: Use a commercial plant/algal DNA extraction kit.
• PCR & Sequencing: Amplify the edited genomic locus using specific primers. Purify PCR products and perform Sanger sequencing.
• Copy Number Variation (CNV) Analysis: For knock-in expression cassettes, perform droplet digital PCR (ddPCR) using primers/probes for the transgene and a reference single-copy endogenous gene. Calculate copy number ratio.
• Off-Target Screening: Use tools like Cas-OFFinder to predict potential off-target sites. Amplify and sequence the top 5-10 predicted sites from long-term cultivated samples.

Data Presentation

Table 1: Phenotypic Stability Metrics Over Serial Passages

Passage Number (≈Generations)	Specific Growth Rate (day⁻¹)	Target Isoprenoid Yield (mg/g DW)	% of Population Expressing Fluorescence Marker
P10 (≈70)	0.45 ± 0.02	5.2 ± 0.3	98
P20 (≈140)	0.44 ± 0.03	5.0 ± 0.4	95
P30 (≈210)	0.43 ± 0.04	4.8 ± 0.5	92
P40 (≈280)	0.42 ± 0.05	4.5 ± 0.6	90
P50 (≈350)	0.41 ± 0.05	4.1 ± 0.7	87
Wild-Type Control	0.46 ± 0.02	0.5 ± 0.1	0

Table 2: Genotypic Analysis of Clonal Offspring at P50

Clone ID	Edit Sequence Integrity (Sanger)	Transgene Copy Number (ddPCR)	Growth Rate (day⁻¹)	Product Yield (mg/g DW)
C1	Intact	1.0	0.42	4.2
C2	Intact	1.1	0.43	4.0
C3	2-bp deletion (heterozygous)	0.5	0.40	2.1
C4	Intact	1.0	0.41	4.3
C5	Silent point mutation	1.0	0.42	4.1
Frequency of Aberration	10% (2/20 clones)	5% (1/20 clones)	N/A	N/A

Visualizations

Title: Genetic Stability Assessment Workflow

Title: Key Genetic Instability Challenges & Analysis Methods

The Scientist's Toolkit: Research Reagent Solutions

Item/Reagent	Function in Stability Assessment
Tris-Acetate-Phosphate (TAP) Medium	Standard liquid and solid culture medium for maintaining and passaging microalgae like C. reinhardtii.
Antibiotic for Selection (e.g., Hygromycin B)	Maintains selective pressure to retain engineered constructs; used in parallel non-selective cultures to test stability.
ddPCR Supermix for Probes (Bio-Rad)	Enables absolute quantification of transgene copy number with high precision, critical for detecting CNV.
Fluorescence-Activated Cell Sorter (FACS)	Instrument for high-throughput isolation of single cells based on fluorescent markers linked to the edit, enabling clonal analysis.
Chloroform:Methanol (2:1 v/v)	Organic solvent mixture for efficient extraction of lipophilic isoprenoid compounds from algal biomass for HPLC/GC-MS.
Plant/Algal Genomic DNA Mini Kit	For high-quality, PCR-ready genomic DNA isolation from tough algal cell walls.
Off-Target Prediction Software (Cas-OFFinder)	In-silico tool to identify potential off-target sites for guide RNAs, informing sequencing-based screening.
Internal Standard (e.g., Nonadecane for GC-MS)	Added to extraction mixtures to allow accurate quantification of target isoprenoid yields.

Within the broader thesis on CRISPR-Cas9 metabolic engineering of Chlamydomonas reinhardtii for high-value isoprenoid production (e.g., astaxanthin, limonene), rigorous evaluation of strain performance is paramount. This application note details the core comparative metrics—titer, yield, productivity, and scaling potential—essential for translating laboratory successes to industrially relevant bioprocesses. These parameters are critical for researchers, scientists, and drug development professionals assessing the economic viability of microalgal platforms.

Core Metrics: Definitions and Context

Titer is the concentration of the target isoprenoid in the fermentation broth at the end of a batch (g/L or mg/L). It indicates the final accumulation capability of the engineered strain.

Yield (often ( Y_{P/S} )) is the mass of product formed per mass of substrate (usually carbon source) consumed (g/g). It reflects metabolic efficiency and carbon flux directed toward the desired pathway.

Productivity is the rate of product formation, typically expressed as volumetric productivity (g/L/day) or specific productivity (g/g cell/day). This metric determines the required bioreactor size and directly impacts capital costs.

Scaling Potential is a composite assessment of how the other three metrics behave during scale-up from flasks to pilot (e.g., 1-10 L photobioreactors) and ultimately to industrial scales. Key factors include robustness to heterogeneous light and nutrient conditions, oxygen transfer, and shear stress.

Table 1: Representative Performance Metrics for Engineered Microalgal Isoprenoid Production

Microalgae Strain	Product	Max Titer (mg/L)	Yield (mg/g DW)	Vol. Productivity (mg/L/day)	Cultivation Scale	Key Engineering Strategy
C. reinhardtii (CC-4348)	Astaxanthin	18.5	4.2	1.54	0.25 L Flask	CRISPRI knockdown of competing carotenoid pathway
C. reinhardtii (CC-503)	Limonene	0.85	0.12	0.043	0.1 L Tube	Heterologous expression of limonene synthase + MEP pathway boost
C. reinhardtii (CC-125)	Bisabolene	1.1	0.18	0.055	0.5 L PBR	Cas9-mediated integration of bisabolene synthase into chloroplast genome
C. reinhardtii (CC-1690)	β-Carotene	25.7	5.8	2.14	1.0 L PBR	Multiplexed Cas9 knockouts of LCYe and HYD genes
C. reinhardtii (CC-124)	Squalene	8.3	1.9	0.69	0.25 L Flask	CRISPRI repression of squalene epoxidase

Experimental Protocols for Metric Determination

Protocol 1: Determining Titer and Yield in Flask Cultures

Objective: Quantify end-point isoprenoid concentration and substrate consumption.

Materials:

• Engineered C. reinhardtii strain.
• TAP (Tris-Acetate-Phosphate) medium with ( ^{13}C )-acetate tracer (for yield calculation).
• 250 mL baffled Erlenmeyer flasks.
• LED incubator with controlled light intensity (e.g., 100 µE/m²/s).
• Centrifuge, freeze-dryer.
• GC-MS or HPLC system for isoprenoid quantification.

Procedure:

• Inoculate 100 mL of TAP medium in triplicate flasks to an initial OD₇₅₀ of 0.1.
• Cultivate at 25°C under continuous light with orbital shaking (120 rpm) for 10-14 days.
• Harvest 10 mL culture daily for OD₇₅₀ (biomass) and media acetate analysis (HPLC).
• At harvest, centrifuge entire culture (4000 x g, 10 min). Wash cell pellet with PBS.
• Freeze-dry pellet to determine Dry Cell Weight (DCW).
• Extract isoprenoids from lyophilized biomass using hexane:ethyl acetate (4:1) with sonication.
• Concentrate extract under nitrogen and resuspend for GC-MS analysis. Quantify using external standard curves.
• Titer Calculation: (Mass of product in extract / Volume of culture harvested) (g/L).
• Yield Calculation (( Y_{P/S} )): (Mass of product / Mass of acetate consumed) (g/g). Acetate consumption = (Initial acetate – Residual acetate).

Protocol 2: Calculating Volumetric Productivity in Photobioreactors (PBRs)

Objective: Measure the rate of product formation in a controlled 1 L flat-panel PBR.

Materials:

• 1 L flat-panel glass PBR with integrated gas sparging and light sensors.
• CO₂-enriched air supply (2% v/v).
• In-line OD probe or manual spectrophotometer.
• Automated sampling system or ports.

Procedure:

• Sterilize PBR and fill with 0.9 L of sterile TAP-S (sulfate-reduced) medium.
• Inoculate with late-exponential phase flask culture to OD₇₅₀ of 0.2.
• Set conditions: 25°C, constant light at 200 µE/m²/s (front illumination), air/CO₂ flow at 0.1 vvm.
• Take 5 mL samples every 12 hours. Measure OD₇₅₀ and cell count.
• Process samples for product quantification as in Protocol 1.
• Volumetric Productivity Calculation: (Titer at time t₂ – Titer at time t₁) / (t₂ – t₁) (g/L/day). Report the maximum observed value during exponential growth.

Protocol 3: Assessing Scaling Potential via Scale-Down Stressors

Objective: Mimic large-scale inhomogeneities in lab-scale cultures to predict scale-up performance.

Materials:

• Multi-zone bioreactor simulator or alternation between high-light and dark flasks.
• Programmable syringe pumps for nutrient pulse/starve cycles.
• Dissolved oxygen (DO) probe.

Procedure:

• Cultivate engineered strain in a 1 L stirred-tank bioreactor under standard conditions to mid-exponential phase.
• Initiate a "scale-down" perturbation protocol for 24-48 hours: a. Light Gradient: Cycle culture between a high-light zone (500 µE/m²/s for 30 sec) and a dark zone (0 µE for 5 min) using a switching apparatus. b. Nutrient Gradient: Periodically spike concentrated acetate to create temporary high-concentration zones, followed by prolonged low-concentration periods.
• Monitor biomass (OD), DO, and product titer throughout.
• Compare final titer, yield, and average productivity to a control culture grown under constant, optimal conditions.
• Scaling Potential Index (SPI): Calculate as (Productivity under stress / Productivity under control) * 100%. Strains with SPI >70% are considered robust.

The Scientist's Toolkit

Table 2: Key Research Reagent Solutions for CRISPR Metabolic Engineering of Microalgae

Item	Function	Example/Supplier
C. reinhardtii Cas9-ready Strain (e.g., UVM4, CC-4533)	High-transformability background strain with minimal silencing.	Chlamydomonas Resource Center (CRC).
CRISPR-Cas9 Ribonucleoprotein (RNP) Complex	For direct delivery of Cas9 protein and sgRNA, reducing off-target integration.	Prepared in-house using recombinant SpCas9 and in vitro transcribed sgRNA.
Gibson Assembly Master Mix	For seamless cloning of donor DNA templates containing pathway genes and homology arms.	New England Biolabs (NEB).
Cell-Penetrating Peptide (CPP) BRP-FP	Facilitates delivery of RNP complexes into algal cells without cell wall removal.	Peptide sequence (BRP-FP: GWTLNSAGYLLGKINLKALAALAKKIL).
TAP-S (-Sulfur) Medium	Enables selective pressure for transformants when using ARS2 or other sulfur-deprivation markers.	Custom formulation per Sueoka (1960).
GC-MS SPME Fiber (e.g., PDMS/DVB)	For headspace sampling of volatile isoprenoids (e.g., limonene) without cell disruption.	Supelco.
Acetate Assay Kit	Enzymatic quantification of acetate consumption for yield (( Y_{P/S} )) calculations.	Megazyme K-ACETRM.
Chloroplast Transformation Kit (for C. reinhardtii)	For integration of pathway genes into the high-copy chloroplast genome.	Bio-Rad PDS-1000/He with 1350 psi rupture discs.

Visualization

CRISPR-Cas9 Metabolic Engineering Workflow

MEP Pathway in Engineered Microalgae

Relationship of Core Bioprocess Metrics

Application Notes

This analysis provides a comparative assessment of microbial chassis—microalgae (e.g., Chlamydomonas reinhardtii, Phaeodactylum tricornutum), yeast (Saccharomyces cerevisiae), and Escherichia coli—for the sustainable production of high-value isoprenoids, a critical focus within CRISPR-Cas9 metabolic engineering research. The selection of an optimal host organism balances sustainability metrics, production economics, and the target product spectrum.

1. Sustainability & Cultivation Microalgae excel in environmental sustainability. They are typically cultivated photoautotrophically, requiring only light, CO₂, and minimal nutrients, leading to a negative carbon footprint. They do not compete with arable land and can utilize wastewater. In contrast, yeast and E. coli are heterotrophic, requiring refined sugar feedstocks (e.g., glucose) derived from agricultural crops, creating a direct land-use and carbon footprint impact. However, heterotrophic fermentation systems offer higher volumetric productivities and easier scale-up in controlled bioreactors.

2. Production Cost & Scalability Cost structures differ significantly. Heterotrophic fermentation with yeast/E. coli has established, predictable scale-up pathways but faces high feedstock costs (~60% of operating costs). Microalgae cultivation, while low in feedstock cost, contends with higher capital expenditure for photobioreactors or open ponds and challenges in achieving consistent, high-density cultures due to light penetration limitations. Downstream processing for intracellular products is similarly complex across all hosts.

3. Product Spectrum & Metabolic Engineering Each host offers distinct advantages for isoprenoid biosynthesis:

• E. coli: Fastest growth, extensive genetic tools, high fluxes through the MEP pathway, but limited native isoprenoid diversity and sensitivity to product toxicity.
• Yeast: Robust, GRAS status, complex eukaryotic compartmentalization (ER, mitochondria) suitable for cytochrome P450-mediated modifications, utilizing the mevalonate (MVA) pathway.
• Microalgae: Native producers of diverse, complex isoprenoids (e.g., carotenoids, phytol); chloroplasts provide an ideal engineered compartment for the MEP pathway with minimal substrate competition. However, genetic toolboxes, though rapidly advancing with CRISPR-Cas9, lag behind model heterotrophs.

4. CRISPR-Cas9 Engineering Context The integration of CRISPR-Cas9 is transformative for microalgae engineering, enabling targeted knockout of competing pathways and precise knock-in of heterologous genes to amplify isoprenoid flux. While routine in yeast and E. coli, its application in microalgae requires species-specific optimization of transformation, Cas9/gRNA delivery, and DNA repair mechanisms (often relying on NHEJ).

Quantitative Comparison Tables

Table 1: Sustainability & Growth Parameters

Parameter	Microalgae (Photoautotrophic)	Yeast (Heterotrophic)	E. coli (Heterotrophic)
Carbon Source	CO₂	Organic C (e.g., Glucose)	Organic C (e.g., Glucose)
Land Use Impact	Low (non-arable)	High	High
Theoretical Max. Biomass (g/L/day)	1-5	50-100	50-100+
Water Recycling	High Potential	Limited	Limited
Typical Cultivation System	PBR/Open Pond	Stirred-Tank Reactor (STR)	Stirred-Tank Reactor (STR)

Table 2: Isoprenoid Production Metrics (Representative)

Product (Class)	Host Organism	Titer (mg/L)	Pathway Engineered	Key Challenge
Lycopene (Carotenoid)	E. coli	3,500	MEP + Heterologous	Toxicity, Redox balance
β-Carotene (Carotenoid)	S. cerevisiae	1,800	MVA + Heterologous	Storage in lipid droplets
Farnesene (Sesquiterpene)	Yarrowia lipolytica	25,000	MVA + Heterologous	Volatility, two-phase extraction
Astaxanthin (Carotenoid)	C. reinhardtii (Engineered)	15	Native MEP enhancement	Low biomass, light regulation

Table 3: Genetic Engineering & CRISPR-Cas9 Efficiency

Aspect	Microalgae	Yeast	E. coli
Transformation Efficiency	Moderate-Low (varies)	High (10⁵-10⁶/µg)	Very High (10⁸-10⁹/µg)
Homology-Directed Repair (HDR) Efficiency	Very Low (NHEJ dominant)	High	Moderate-High
CRISPR-Cas9 Tool Maturity	Emerging, species-specific	Well-established	Well-established
Standardized Parts (Promoters, etc.)	Limited, developing	Extensive	Extensive

Experimental Protocols

Protocol 1: CRISPR-Cas9-Mediated Gene Knockout in C. reinhardtii for MEP Pathway Flux Enhancement Objective: Disrupt a competing pathway gene (e.g., LYC for lycopene cyclase) to shunt flux towards target linear isoprenoids.

• gRNA Design & Construct Assembly: Design two 20-nt guide RNAs targeting exonic regions of the target gene. Clone expression cassettes for each gRNA and a codon-optimized Cas9 (with nuclear/chloroplast targeting as needed) into a microalgae-specific binary vector using Golden Gate assembly.
• C. reinhardtii Transformation (Glass Bead Method): Harvest mid-log phase CC-125 cells, wash, and concentrate to 2x10⁸ cells/mL in TAP + 40 mM sucrose. Mix 300 µL cells with 10 µg linearized plasmid DNA and 0.3 g sterile glass beads (425-600 µm). Vortex at max speed for 15 sec. Plate immediately onto TAP + sucrose agar. After 24h, transfer to selective agar (e.g., paromomycin).
• Mutant Screening: After 7-10 days, pick colonies. Isolate genomic DNA. Perform PCR amplification of the target locus (~1.5 kb). Analyze products via agarose gel for size shifts. Confirm by Sanger sequencing of PCR products to identify indels.
• Phenotypic Validation: Grow putative mutants in TAP medium under moderate light. Extract pigments (using acetone:methanol 7:3) and analyze via HPLC for carotenoid/isoprenoid profile changes.

Protocol 2: Batch Fermentation for Sesquiterpene Production in Engineered S. cerevisiae Objective: Assess isoprenoid titer and yield in a controlled bioreactor.

• Seed Culture: Inoculate a single colony of engineered yeast into 50 mL of SD-URA medium in a 250 mL baffled flask. Incubate at 30°C, 250 rpm for 16-18 h.
• Bioreactor Setup & Inoculation: Sterilize a 2 L bioreactor containing 1 L of defined fermentation medium (e.g., SM + 2% glucose). Calibrate pH and DO probes. Inoculate at OD600 of 0.1. Set parameters: 30°C, pH 5.5 (controlled with NH₄OH), DO >30% (via agitation/aeration cascade).
• Fed-Batch Operation: Allow initial batch phase until glucose depletion (DO spike). Initiate glucose feed (500 g/L) at a constant rate to maintain low, non-repressing levels. Continue fermentation for 72-96 h.
• Sampling & Analysis: Take periodic samples for OD600, glucose (HPLC-RID), and product quantification. For hydrophobic sesquiterpenes (e.g., farnesene), add a 10% dodecane overlay for in situ extraction. Analyze overlay via GC-MS.

Protocol 3: Chloroplast Transformation in Phaeodactylum tricornutum for Heterologous Gene Expression Objective: Express a plant-derived sesquiterpene synthase in the diatom chloroplast.

• Vector Design: Clone the gene of interest, flanked by diatom chloroplast homology arms (targeting the psbA or rbcL locus) and driven by a chloroplast promoter (e.g., psbA), into a pUC-based vector.
• Biolistic Transformation: Coat 0.6 µm gold microparticles with 5 µg of plasmid DNA. Spread late-log phase P. tricornutum cells on f/2 + 1% agar plates. Use a helium-driven gene gun (1,100 psi rupture disk, 6 cm target distance) to bombard plates.
• Selection & Homoplasmy: Incubate under light for 24h, then transfer cells to f/2 + 1% agar plates containing spectinomycin (100 µg/mL). Restreak resistant colonies every 3-4 weeks for 3 months to achieve homoplasmy.
• Confirmation: Isolate total DNA. Perform PCR with primers external to the homology region to confirm integration. Use Southern blotting to confirm homoplasmy.

Diagrams

CRISPR Workflow & Host Selection Logic

MEP & MVA Pathways to Isoprenoids

The Scientist's Toolkit

Table 4: Key Research Reagent Solutions for Microalgae Metabolic Engineering

Reagent / Material	Function in Research	Example Product / Vendor
CRISPR-Cas9 Expression Vector (Microalgae-optimized)	Delivers Cas9 and gRNA(s) to the host cell; contains species-specific regulatory elements and selection marker.	pChlamy-CAS9, pPhaeo-CAS9 (Addgene).
Microalgae-Specific Cell Wall-Deficient Strain	Facilitates higher transformation efficiency by eliminating the physical barrier to DNA uptake.	C. reinhardtii cw15 mutant (CC-503).
Golden Gate Assembly Kit	Enables rapid, modular assembly of multiple genetic parts (promoters, genes, terminators) into the destination vector.	MoClo Toolkit for Microalgae.
Biolistic Transformation Kit	Delivers DNA-coated microparticles into cells with rigid walls (e.g., diatoms) via high-velocity propulsion.	Bio-Rad PDS-1000/He System, Gold Microcarriers.
TAP / f/2 Culture Media Kits	Standardized, consistent nutrient sources for axenic microalgal cultivation in lab settings.	Tris-Acetate-Phosphate (TAP) Medium, Guillard's f/2 Medium.
Isoprenoid Extraction Solvent	Efficiently lyses cells and solubilizes hydrophobic isoprenoid products for downstream analysis.	Acetone:Methanol (7:3 v/v) or Hexane:Ethyl Acetate.
HPLC/GC-MS Standards	Authentic chemical standards for quantifying and identifying isoprenoid products via chromatography.	β-carotene, Farnesene, Limonene (Sigma-Aldrich).
Spectinomycin / Paromomycin	Selective antibiotics for transformant selection in microalgae and chloroplast engineering.	Thermo Fisher Scientific.

Application Notes

Within the context of metabolic engineering of microalgae for isoprenoid production via CRISPR-Cas9, the selection of a production chassis requires a critical evaluation of upstream cultivation and downstream processing. These notes compare microalgae and traditional plant systems across three pivotal parameters.

1. Land and Resource Use Microalgae cultivation systems (photobioreactors, PBRs; open ponds) demonstrate a radical reduction in land footprint compared to terrestrial crop cultivation. This is quantified as Annual Biomass Yield per Hectare. Furthermore, engineered microalgae strains can utilize non-arable land and saline/brackish water, presenting no competition with food crops. In contrast, cultivating high-yield medicinal plants (e.g., Artemisia annua for artemisinin) requires fertile arable land, significant freshwater inputs, and is subject to seasonal and climatic variability, jeopardizing stable biomass supply for industrial extraction.

2. Process Controllability and Engineering Potential Controllability is paramount for consistent, high-titer metabolite production. Enclosed PBRs offer fine-tuned control over temperature, pH, light intensity/duration, nutrient supply, and gas exchange (CO₂/O₂), enabling optimization of growth and metabolic flux towards target isoprenoids. This controlled environment is essential for inducing engineered pathways in CRISPR-edited strains. Plant cultivation in open fields is subject to uncontrollable biotic (pests, pathogens) and abiotic (drought, frost) stresses, leading to batch-to-batch variability in metabolite content. The genetic tractability of microalgae, especially model species like Chlamydomonas reinhardtii and Phaeodactylum tricornutum, far exceeds that of most plants, allowing for precise CRISPR-Cas9-mediated knockout/knock-in of genes in the MEP/DOXP isoprenoid pathways.

3. Extraction Efficiency and Downstream Processing Extraction efficiency is a function of biomass pre-treatment, solvent use, and target molecule accessibility. Microalgae biomass, with less complex and rigid cellular structures (lacking lignin), often requires less harsh pre-treatment, facilitating cell disruption and solvent penetration. The homogeneity of microalgae biomass leads to more predictable extraction yields. Plant tissues are heterogeneous (roots, leaves, bark) and contain complex polymers (lignin, cellulose), necessitating more energy-intensive and often compound-specific pre-treatment steps, which can degrade thermolabile isoprenoids.

Table 1: Quantitative Comparison of Land Use and Production Parameters

Parameter	Microalgae (PBR)	Microalgae (Open Pond)	Medicinal Plants (e.g., Artemisia annua)
Land Use (ha/yr per kg biomass)	0.001 - 0.01	0.02 - 0.05	0.5 - 2.0
Water Consumption (L/kg biomass)	250 - 500	500 - 1000	5000 - 20,000
Annual Biomass Yield (tons DW/ha/yr)	50 - 150	20 - 50	1 - 10
Growth Cycle (time to harvest)	3 - 10 days	5 - 15 days	3 - 8 months
Controllability (Environmental)	Very High	Low-Moderate	Very Low
Genetic Tractability for Engineering	Very High	Very High	Low-Moderate

Table 2: Comparison of Extraction Efficiency Metrics for Isoprenoids

Parameter	Microalgae Biomass	Plant Biomass (Leaf Tissue)
Typical Cell Disruption Method	Sonication, Bead Milling, High-Pressure Homogenization	Freeze-Drying & Grinding, Steam Distillation
Required Pre-treatment Severity	Low-Moderate	High
Solvent Consumption (L/kg DW)	10 - 30	20 - 50
Extraction Time (hours)	1 - 6	6 - 24
Theoretical Yield Variance	Low (Homogeneous biomass)	High (Tissue, season, genotype variance)

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Experimental Protocols

Protocol 1: Cultivation and Harvesting of CRISPR-Edited Microalgae for Isoprenoid Analysis Objective: Generate biomass from engineered strains under controlled conditions for downstream metabolite extraction.

• Strain & Medium: Inoculate CRISPR-Cas9-edited C. reinhardtii strain (e.g., engineered for GGPP overproduction) in TAP medium with appropriate selection antibiotics.
• Cultivation: Grow in a multi-cultivator PBR system at 25°C, under continuous light (150 μmol photons m⁻² s⁻¹), with 2% CO₂-enriched air bubbling. Monitor OD₇₅₀ daily.
• Harvest: During mid-log phase (OD₇₅₀ ~1.5), concentrate cells via centrifugation at 4,000 x g for 10 min at 4°C.
• Biomass Processing: Wash pellet twice with fresh medium or buffer. For metabolite analysis, immediately flash-freeze pellet in liquid N₂ and lyophilize for 48h. Store dried biomass at -80°C.

Protocol 2: Simultaneous Extraction and Quantification of Isoprenoids from Microalgae/Plant Biomass Objective: Efficiently extract and quantify terpenoid molecules (e.g., carotenoids, sesquiterpenes) from dried biomass.

• Cell Disruption:
  • Microalgae: Weigh 50 mg lyophilized powder. Add 1 mL extraction solvent (e.g., 9:1 Methanol:DMSO with 0.1% BHT). Homogenize using a bead beater with 0.5mm zirconia beads for 3 cycles of 1 min with 1 min cooling on ice.
  • Plant Tissue: Weigh 100 mg lyophilized, ground leaf powder. Add 1.5 mL of the same extraction solvent. Sonicate in an ice bath for 15 min (pulse: 30s on, 30s off).
• Extraction: Incubate samples in the dark at 55°C for 30 min with occasional vortexing.
• Clarification: Centrifuge at 15,000 x g for 10 min at 4°C. Collect supernatant.
• Re-extraction: Re-suspend pellet in 0.5 mL fresh solvent, repeat steps 2-3. Pool supernatants.
• Analysis: Filter through a 0.22 μm PTFE syringe filter. Analyze via RP-HPLC or LC-MS/MS using appropriate standards (e.g., β-carotene, artemisinic acid). Use a C18 column and a gradient of water/acetonitrile.

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Pathway and Workflow Visualizations

Title: CRISPR Workflow for Microalgae Isoprenoid Engineering

Title: Biomass to Extract: Microalgae vs Plant Workflow

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The Scientist's Toolkit: Research Reagent Solutions

Item	Function in Context	Example Application
CRISPR-Cas9 Kit for Microalgae	Delivery of ribonucleoprotein (RNP) complexes for gene editing. Enables knockout of competing pathways (e.g., phytone synthase) in the carotenoid branch.	Chlamydomonas RNP Transfection Kit.
Photobioreactor (Multicultivator)	Provides controlled, parallel, small-scale cultivation for strain screening under defined light, temperature, and gas conditions.	PSI Multicultivator MC-1000.
High-Pressure Homogenizer	Efficient mechanical disruption of robust microalgal cell walls (e.g., Nannochloropsis) to release intracellular isoprenoids.	Avestin EmulsiFlex-C5.
Bead Beater Homogenizer	Rapid mechanical lysis of microalgae cells using grinding beads, ideal for small-volume, high-throughput sample preparation.	Bertin Instruments Precellys.
Lyophilizer (Freeze Dryer)	Removes water from biomass under low temperature/pressure, preserving thermolabile isoprenoids and stabilizing biomass for storage.	Labconco FreeZone.
Sonication Probe	Applies ultrasonic energy to disrupt cell membranes and enhance solvent penetration in both plant and algae samples.	Qsonica Q700.
Solid-Phase Extraction (SPE) Cartridges	Post-extraction clean-up and concentration of target isoprenoids from crude solvent extracts prior to analytical quantification.	C18 or Diol-phase SPE columns.
Isoprenoid Analytical Standards	Essential reference compounds for calibrating HPLC/LC-MS systems to identify and quantify specific engineered metabolites.	β-carotene, Farnesol, Artemisinin.

Techno-Economic Analysis (TEA) and Life Cycle Assessment (LCA) for Feasibility Studies

Application Notes

The integration of Techno-Economic Analysis (TEA) and Life Cycle Assessment (LCA) is critical for evaluating the commercial and environmental viability of metabolic engineering projects. Within the thesis context of using CRISPR-Cas9 to engineer microalgae (e.g., Chlamydomonas reinhardtii, Nannochloropsis spp.) for high-value isoprenoid (e.g., β-carotene, astaxanthin, squalene) production, these analyses provide a dual-perspective framework. TEA quantifies the cost drivers and revenue potential at scale, while LCA maps the environmental impacts from a cradle-to-gate perspective, identifying sustainability trade-offs of genetic modifications, cultivation, and downstream processing. This holistic feasibility study is essential for guiding research priorities towards economically sound and environmentally sustainable bioprocesses for pharmaceutical and nutraceutical applications.

Table 1: TEA Cost Drivers for Microalgae-Based Isoprenoid Production (Model at 10,000 L scale)

Cost Category	Key Components	Estimated Contribution to Operating Cost (%)	Notes for CRISPR-Cas9 Engineered Strain
Capital Costs	Photobioreactors, Harvesting (Centrifugation), Extraction Equipment	25-40%	Higher upfront cost for sterile, controlled PBRs vs. open ponds.
Cultivation Media	Nutrients (N, P, Trace Metals), Carbon Source (CO2)	20-30%	Engineered strains may require specific supplements or have reduced nutrient demands.
Operating Labor	Monitoring, Inoculation, Maintenance	10-15%	Similar to wild-type cultivation.
Harvesting & Dewatering	Centrifugation, Flocculation, Filtration	15-25%	Biomass concentration and cell wall modifications via CRISPR can impact efficiency.
Extraction & Purification	Cell Disruption, Solvent Extraction, Chromatography	20-35%	Isoprenoid yield and intracellular location significantly affect cost. Target compound purity (>95%) for pharma increases cost.
Utilities	Lighting, Sterilization, Cooling, Mixing	5-10%	LED lighting efficiency is crucial. Engineered strains with higher photosynthetic efficiency reduce cost.

Table 2: LCA Impact Indicators for Microalgae Isoprenoid Pathways (Per kg of Product)

Impact Category	Units	Conventional Extraction (Baseline)	CRISPR-Optimized Strain (Projected)	Primary Driver for Difference
Global Warming Potential	kg CO2-eq	50-120	30-80	Energy source for PBR operation and solvent use in extraction.
Water Consumption	m³	800-2000	600-1500	Evaporation losses in open ponds; closed PBRs and reduced cultivation time improve metrics.
Fossil Resource Scarcity	kg oil-eq	20-50	15-40	Fossil-based electricity and chemicals (solvents, fertilizers).
Land Use	m²a crop eq	10-30	8-25	Increased volumetric productivity reduces land footprint.

Experimental Protocols

Protocol 1: Gate-to-Gate TEA for Microalgae Isoprenoid Bioprocess

Objective: To model the manufacturing cost per gram of purified isoprenoid from a CRISPR-engineered microalgae strain.

Methodology:

• Process Design & Scaling: Define a base case process flow diagram (PFD). Key unit operations include: Inoculum Prep → Cultivation (Photobioreactor) → Harvesting (Flocculation + Centrifugation) → Cell Disruption (Bead Milling) → Extraction (Supercritical CO2 or Ethanol) → Purification (Chromatography).
• Mass & Energy Balance: Using experimental data (yield: mg/g DW, productivity: mg/L/day), perform mass balances for 1 kg of final product. Calculate energy inputs for mixing, lighting, cooling, and downstream processing.
• Capital Cost Estimation (CAPEX): Use factored estimation. Obtain quotes for major equipment (PBR, centrifuge, extractor). Multiply by Lang factors (3-5) for total installed plant cost. Annualize using a capital charge factor (e.g., 0.15-0.20/yr).
• Operating Cost Estimation (OPEX): Itemize:
  • Raw Materials: Quantify culture media, CO2, solvents.
  • Utilities: Model electricity, water, and cooling demands from Step 2.
  • Labor: Estimate full-time equivalents for operations.
  • Waste Disposal: Cost for spent biomass and solvent recovery.
• Financial Analysis: Calculate the minimum product selling price (MSP) at a target return on investment (e.g., 10-20%). Perform sensitivity analysis on key parameters: isoprenoid yield, growth rate, extraction efficiency, and energy cost.

Protocol 2: Cradle-to-Gate LCA for Engineered Microalgae Product

Objective: To evaluate the environmental impacts associated with producing 1 kg of isoprenoid from a CRISPR-engineered strain compared to a baseline.

Methodology:

• Goal & Scope Definition: Functional Unit: 1 kg of purified isoprenoid (≥95% purity). System Boundary: Cradle-to-Gate (includes production of inputs, cultivation, harvesting, extraction, purification. Excludes distribution, use, end-of-life).
• Life Cycle Inventory (LCI):
  • Foreground System: Collect primary data from lab-scale protocols: nutrient consumption, electricity for PBR lighting/mixing, water use, solvent volumes, and yields.
  • Background System: Use commercial LCA databases (e.g., Ecoinvent) to model impacts of upstream processes (e.g., fertilizer production, electricity grid mix, solvent manufacture).
• Life Cycle Impact Assessment (LCIA): Select relevant impact categories (Table 2). Calculate characterization factors using a method like ReCiPe 2016. Key categories include Global Warming Potential (GWP), Water Consumption, and Fossil Resource Scarcity.
• Interpretation & Hotspot Analysis: Identify processes contributing >60% to each impact category. Compare the CRISPR-engineered scenario to a baseline (wild-type strain or chemical synthesis). Uncertainty analysis via Monte Carlo simulation is recommended.

Visualizations

TEA and LCA Parallel Workflow for Feasibility

CRISPR Engineering Impacts on TEA and LCA Metrics

The Scientist's Toolkit: Research Reagent Solutions

Item	Function in CRISPR Microalgae/Isoprenoid Research	Example/Brand Considerations
CRISPR-Cas9 System	Enables targeted knock-out/knock-in of genes in the MEP pathway or regulatory factors.	Alt-R CRISPR-Cas9 System (IDT); custom gRNA design for microalgae genomes.
Microalgae Strain	Photosynthetic chassis for isoprenoid production. Requires transformability.	Chlamydomonas reinhardtii (CC-125), Nannochloropsis oceanica (IMET1).
Electroporator / Gene Gun	For delivery of CRISPR constructs into microalgae cells.	Bio-Rad Gene Pulser or PDS-1000/He System.
HPLC-MS/MS	Quantification of specific isoprenoid products (e.g., astaxanthin, β-carotene) from cell extracts.	Agilent 1290 Infinity II/6470 system; C30 carotenoid columns.
Photobioreactor (Lab-scale)	Provides controlled, reproducible cultivation conditions for growth and yield experiments.	DASGIP or INFORS HT Multifors systems with LED lighting.
Supercritical Fluid Extractor (SFE)	Green technology for efficient, solvent-minimized extraction of lipophilic isoprenoids.	Waters Thar SFC or lab-scale SFE systems using CO2.
LCA Software & Database	To model environmental impacts based on experimental inventory data.	SimaPro or OpenLCA software with Ecoinvent database.
TEA Modeling Software	To build process models and perform cost sensitivity analyses.	ASPEN Plus, SuperPro Designer, or customized Excel models.

Conclusion

CRISPR-Cas9 has unequivocally transformed microalgae into programmable, solar-powered cell factories for isoprenoid production. This guide has outlined the journey from foundational pathway understanding through precise genome editing, yield optimization, and rigorous strain validation. The key takeaway is that while challenges in transformation efficiency and metabolic burden persist, integrated systems biology and advanced engineering strategies are rapidly overcoming these hurdles. Compared to heterotrophic microbes and traditional agriculture, engineered microalgae offer a compelling, sustainable alternative with a reduced carbon footprint. For biomedical research, this technology paves the way for a reliable, scalable supply of complex terpenoid precursors for drug discovery (e.g., artemisinin, taxol analogs) and high-purity nutraceuticals. Future directions must focus on developing universal genetic tools for diverse algal species, engineering secretion mechanisms, and integrating biorefining concepts to maximize economic viability, ultimately accelerating the transition from lab innovation to clinical and commercial reality.