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

• Published in: Cell reports methods 2023-08, Vol.3 (8), p.100562-100562, Article 100562
• Main Authors: Nievergelt, Adrian Pascal, Diener, Dennis Ray, Bogdanova, Aliona, Brown, Thomas, Pigino, Gaia
• Format: Article
• Language: English
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2023.100562

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 demonstrate how to use this pipeline to facilitate the generation of mutant lines without residual selectable markers by co-targeted insertion. Finally, we describe how insertional cassettes can be erroneously mutated during insertion and suggest strategies to select for lines that are modified as designed. • CRISPR-Cas9 allows endogenous tagging of Chlamydomonas genes • High-throughput crossing reduces the time to combine mutant traits • An optimized PCR/qPCR protocol allows robust genotyping of crude extracts • Insertional mutagenesis is impeded by microhomology-driven fragment exchange The ability to perform precise genome editing is an indispensable tool in the study of any model organism. In Chlamydomonas , a long-established model organism for study of genetics, cilia motility, and photosynthesis, homology-directed precision editing has been considered difficult. Here, we present a CRISPR-Cas ribonucleoprotein-based method for homology-directed knockin mutagenesis of Chlamydomonas and demonstrate that the approach can produce endogenously tagged cell lines in a matter of weeks. Nievergelt et al. present a CRISPR-Cas-mediated knockin approach for precision genome editing in Chlamydomonas . The work also generates rapid crossing and robust high-throughput genomic screening strategies and provides a toolbox for the Chlamydomonas community to rapidly produce the precise mutant line required for any given study.