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

BACKGROUND: Chlamydomonas reinhardtii P.A. Dang. is a model organism for studying the genetics of green algae. To expand and improve the genetic engineering toolkit for microalgae, we used the binary plant vector pKSE401, based on the CRISPR/Cas9 system, to knock out the PSY1 (PHYTOENE SYNTHASE 1) gene in C. reinhardtii, which encodes a key enzyme in the metabolic pathway of carotenoid biosynthesis. Mutations in this gene result in the appearance of a characteristic white or pale-green colony phenotype, which allows the selection of transformants with disruptions in the PSY1 gene based on phenotype.

AIM: To evaluate the feasibility of using the binary plant vector pKSE401 for CRISPR/Cas9-mediated gene knockout in the microalga C. reinhardtii.

METHODS: The vector pKSE401-PSY1 was constructed containing a previously used guide RNA spacer targeting the PSY1 gene of C. reinhardtii. Two wild-type strains were used in this study: CC-124 (wt, mt–) and 137c (wt, mt+). Experiments were performed in three biological and three technical replicates. Cell cultivation and transformation conditions (electroporation method), as well as the screening protocol for psy1 transformants (white/pale-green colony phenotype as a selection system, PCR and sequencing for verification of genome editing), were carried out according to published protocols.

RESULTS: A total of 164 transformant colonies were obtained and analyzed, of which 29 displayed the white or pale-green phenotype (17.7%). Sequencing confirmed the presence of insertions/deletions in the target site of the PSY1 gene in 13 mutants, whereas PCR amplification failed for three mutants. The overall efficiency of targeted editing of the PSY1 gene (across all experimental variants) reached 7.9%.

CONCLUSION: The preliminary results demonstrate the feasibility of using the plant binary vector pKSE401 for gene knockout in the green alga C. reinhardtii, thereby expanding the range of potential target species for its application. The system has substantial limitations (random plasmid integration into the genome and relatively low editing efficiency). However, further optimization of the protocol may help overcome some of these limitations.