TABLE 1.
Examples of CRISPR-based bioengineering in different microalgal species to produce industrially important biomolecules.
| Strain | Target gene modified | Type of modification | Type of nuclease | Outcomes | References |
|---|---|---|---|---|---|
| Chlamydomonas reinhardtii | |||||
| CC-4349 | CpFTSY, zeaxanthin epoxidase (ZEP) | Knockout | Cas9 | Mutant strain capable of constitutively producing 13-fold zeaxanthin with improved photosynthetic productivity, greater biomass accumulation observed under high light growth conditions | Baek et al. (2016) |
| CC-400 | phosphoenolpyruvate carboxylase isoform 1 (CrPEPC1) | CRISPRi | dCas9 | CrPEPC1 downregulated strains showed increase in lipid content by 28.5% dry cell weight basis compared to WT | Kao and Ng (2017) |
| CC-4349 | zeaxanthin epoxidase (ZEP) | Knockout | Cas9 | Mutant strain exhibited significantly higher zeaxanthin content (56-fold) and productivity (47-fold) than the WT without the reduction in lutein level | Baek et al. (2018) |
| CC-4349 | phospholipase A2 (PLA2) | Knockout | Cas9 | Lipid productivities of phospholipase A2 knockout mutants increased by approximately 64.25% | Shin et al. (2019) |
| CC-4349 | ELT | Knockout | Cas9 | Higher lipid accumulation in mutant strain compared to WT and noticeable shift in fatty acid composition with an increase of approximately 27.2% in the C18:1 proportion making it suitable for biofuel production | Nguyen et al. (2020) |
| CC-4349 | Zeaxanthin epoxidase (ZEP), lycopene epsilon cyclase (LCYE) | Knockout | Cas9 | Zeaxanthin yield of double knockout mutant (ZEP and LCYE) strains were 60% higher. Zeaxanthin yield reported was (5.24 mg/L) compared to single ZEP mutant | Song et al. (2020) |
| CC-4349 | ADP-glucose pyrophosphorylase (AGP), zeaxanthin epoxidase (ZEP) | Knockout | Cas9 | Double knockout strains (AGP and ZEP) demonstrated 81% higher oil productivity along with same zeaxanthin and lutein concentrations as that of WT | Song et al. (2022) |
| UVM11 | Bacterial phytase gene | knockin | Cas9 | Showed site specific integration of bacterial phytase gene which clearly demonstarted that complex recombinant proteins and novel synthetic biomolecules can be successfully produced in food grade organism C. reinhardtii | Zadabbas Shahabadi et al. (2023) |
| Phaeodactylum tricornutum | |||||
| CCMP2561 | CryP | Knockout | Cas9 | Knockout showed increase in fucoxanthin content by 1.29-fold compared to WT | Yang et al. (2022) |
| Pt1 wild type (WT) | ptACSL3 | Knockout | Cas9 | PtACSL3 mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1 | Hao et al. (2022) |
| CCAP 1055/1, & CCMP2712 | Violaxanthin de-epoxidase (VDL2) and zeaxanthin epoxidase (ZEP1) | Knockout | Cas9 | Demonstrated the role of two genes in Fucoxanthin biosynthesis. Knockout mutants of VDL2 and ZEP1 did not produce fucoxanthin | Bai et al. (2022) |
| P. tricornutum Bohlin | enoyl CoA hydratase (PtECH) | CRISPRi | dCas9 | The ECH knockdown mutants exhibited an enhanced lipid accumulation relative to WT. The mutants showed higher photosynthetic efficiency, but impaired growth compared to WT | Guo et al. (2023) |
| Nannochloropsis spp. | |||||
| N. gaditana CCMP1894 | ZnCys | Attenuation and Knockout | Cas9 | Attenuated mutants of ZnCys demonstrated double the strain’s lipid productivity (∼5.0 g/m2/d) compared to WT (∼2.5 g/m2/d) and retained the ability to grow and fix CO2 at levels nearly equivalent to those of the WT strain | Ajjawi et al. (2017) |
| N. gaditana CCMP1894 | Acyl-CoA oxidase (Aco1) | Knockout and Knockin | Cas9 | Knockout mutant strains showed double the lipid productivity compared to WT | Verruto et al. (2018) |
| N. salina CCMP1776 | Three cellulose synthase genes (cesA1, cesA2, cesA4) | Knockout | Cas9 | Cell wall thickness and cellulose content were reduced in cesA1 mutant but not in cesA2 or cesA4 cells. CesA1 mutation resulted in a reduction of chrysolaminarin and neutral lipid contents, by 66.3% and 37.1%, respectively, but increased the soluble protein content by 1.8-fold. Thinned cell wall cells were susceptible to mechanical stress, resulting in a 1.7-fold enhancement of lipid extractability | Jeong et al. (2020) |
| N. gaditana CCMP526 | beta-glucan synthase (BGS), transglycosylase (TGS) | Knockout | Cas9 | The generated knockout mutants showed ∼5-fold lower accumulation of soluble carbohydrate (β-1,3-glucose oligomers) following nitrogen starvation compared to WT, without any observed growth defect. The TGS knockout mutants showed 25%–40% (dry cell weight) increases in total fatty acids | Vogler et al. (2021) |
| N. salina CCMP1776 | Δ12-fatty acid desaturase (FAD12) | Knockin | Cas9 | The targeted knockin mutants of FAD12 showed four-fold higher production of linoleic acid and 1.5-fold increase in eicosapentaenoic acid | Ryu et al. (2021) |
| N. oceanica IMET1 | bZIP-family regulator NobZIP77 | Knockout | Cas9 | The NobZIP77 knockout mutants under nitrogen deprivation showed similar growth like WT, with 3 times more triacylglycerol productivities | Zhang et al. (2022) |
| Chlorella spp. | |||||
| C. vulgaris FSP-E | Omega-3 fatty acid desaturase (fad3) | Knockout | Cas9 | Knockout mutants of fad3 showed 46% (w/w) higher lipid content over WT | Lin and Ng (2020) |
| C. sorokiniana UTEX 1602 | — | CRISPRa/i [Adaptive Single Guide Assisted Regulation DNA (ASGARD)] | dCas9 | Gene regulation via CRISPRa-VP64 (CRISPRa) enhanced the protein contents up to 60% (w/w) of dry cell weight, where the highest concentration was 570 mg/L, while CRISPRi-KRAB (CRISPRi) with ASGARD increased protein content to 65% and lipid formed in the range of 150–250 mg/L (WT: 150 mg/L) | Lin et al. (2022) |
| Parachlorella kessleri NIES-2152 | Three genes, calcium-dependent membrane targeting (9934_t), duplicated mannanases 1 (8741_t), plastidic ATP/ADP translocase (9067_t) | Knockout | Cas9 | The knockout mutants of plastidic ATP/ADP translocase showed >30% more lipid productivity compared to WT strain under dark light cycle | Kasai et al. (2023) |
| Tetraselmis spp. | |||||
| Tetraselmis spp. KCTC12432BP | ADP-glucose pyrophosphorylase (AGP) | Knockout | Cas9 | AGP mutants showed enhanced lipid production. Fatty acids in the AGP mutants increased by 274% and 314% compared to WT. The mutants showed 2.3 and 2.7-fold higher lipid productivity than the WT | Chang et al. (2020) |
| Porphyridium purpureum | |||||
| P. purpureum CCMP 1328 | Chlorophyll synthase (CHS1) | Knockout | Cas9 | CHS1 mutants produced significantly higher phycoerythrin compared to WT | Jeon et al. (2021) |