Efficient precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.

ABSTRACT: CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line. We also present methods for high-throughput crossing of transformants and a custom quantitative PCR (qPCR)-based high-throughput screening of mutants as well as meiotic progeny. We demo

SUBMITTER: Nievergelt AP 

PROVIDER: S-EPMC10475843 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature