CRISPR/Cas based genome editing in microalgae

Abstract

CRISPR/Cas systems are presently the most attractive genome editing technology, that is widely used for genetic engineering of various crops and industrial microorganisms. Currently, application of the CRISPR/Cas based genome editing promises advances in microalgae biotechnology aimed at boosting the output of biofuels and valuable bioactive compounds. However, algae remain relatively complex objects for genetic manipulation [1]. The main problems are associated with the need of a species-oriented approach when creating a transformation toolbox due to the peculiarities in the structure of membranes and the cell wall of a particular taxon. The proper selection and design of a CRISPR construct is also required due to the possible presence of a powerful silencing system against introduced genetic constructs in the cell. These difficulties explain the low efficiency of microalgae transformation and the meager list of successfully edited species [1, 2].

The first instance of genome editing in microalgae using CRISPR/Cas was reported in Chlamydomonas reinhardtii P.A. Dang [3]. To date, four transformation methods (Agrobacterium-mediated, particle bombardment, glass beads agitation, electroporation) have been successfully used for editing (knock-in and knock-out) the C. reinhardtii genome with two types of CRISPR constructs (plasmid and ribonucleoprotein). The developed protocols make it possible to achieve high efficiency of genomic editing — for example, in our study it varied from 10.6% to 68.8% [4]. These benefits along with completely sequenced genome, well-studied genetics, accessibility and haplontic life cycle makes C. reinhardtii an outstanding model organism for CRISPR/Cas application in microalgae research [5].

Keywords

CRISPR/Cas / genome editing / transformation toolbox / CRISPR construct delivery / microalgae / GMOs

References

[1] Jeon S, Lim J-M, Lee H-G, et al. Current status and perspectives of genome editing techno-logy for microalgae. Biotechnology for Biofuels. 2017;10:267. DOI: 10.1186/s13068-017-0957-z

[2] Patel VK, Das A, Kumari R, Kajla S. Recent progress and challenges in CRISPR-Cas9 engineered algae and cyanobacteria. Algal Research. 2023;71. DOI: 10.1016/j.algal.2023.103068

[3] Jiang W, Brueggeman AJ, Horken KM. Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryotic Cell. 2014;13(11): 1465–1469. DOI: 10.1128/ec.00213-14

[4] Virolainen PA, Chekunova EM. Optimization of CRISPR/Cas9 method for transgenesis of model microalgae Chlamydomonas reinhardtii [abstract]. Ecological genetics. 2022;20(Suppl. 1): S42–43. DOI: 10.17816/ecogen112332

[5] Naduthodi MIS, Barbosa MJ, van der Oost J. Progress of CRISPR-Cas based genome editing in photosynthetic microbes. Biotechnology Journal. 2018;13(9). DOI: 10.1002/biot.201700591

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