Application of the CRISPR/Cas9 pKSE401 vector for knockout of the PSY1 gene in green alga Chlamydomonas reinhardtii: a brief report on the first results

BACKGROUND: Chlamydomonas reinhardtii P.A.Dang. – a model object for studying the genetics of green algae. In order to expand and improve the tools for genetic engineering of microalgae, we applied the CRISPR/Cas9 plant binary vector pKSE401 for knockout of the gene PSY1 (PHYTOENE SYNTHASE 1) encoding a key enzyme in the metabolic pathway of carotenoid biosynthesis in C. reinhardtii. Mutations in this gene provide a convenient phenotype-based selection system (white/pale green colonies).

AIM: The aim of our study was to examine the possibility of using the pKSE401 plant binary vector for CRISPR/Cas9-mediated gene knockout in microalga C. reinhardtii.

METHODS: We created the pKSE401-PSY1 vector, which carries a previously applied guide RNA spacer to the PSY1 gene of C. reinhardtii. Wild-type strains were used in the work: CC-124 (wt, mt-) and 137c (wt, mt+). The experiments were carried out in three biological and three technical repetitions. Cell culture and electroporation conditions, as well as screening protocol of psy1 transformants (white/pale green colony coloration as a selective system, PCR and sequencing for verification), were carried out in accordance with the published protocols.

RESULTS: As a result, 164 colonies of transformants were obtained and analyzed, of which 29 had a white/pale green phenotype (17.7%). Of these, 13 mutants were confirmed to have insertions/deletions in PSY1 gene target site by sequencing, and for 3 mutants we failed to generate a PCR product. The overall effectiveness of targeted editing of the PSY1 gene (in all experimental variants) reached 7.9%.

CONCLUSION: The preliminary results obtained demonstrate the possibility of using the plant binary vector pKSE401 to knockout genes in green alga C. reinhardtii, expanding the range of possible target species for its application. The system used has significant limitations (random plasmid insertion into the genome, low gene editing efficiency), but further improvement of the protocol may eliminate several of the identified shortcomings.