Protocol for precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas

• Published in: STAR protocols 2024-03, Vol.5 (1), p.102774-102774, Article 102774
• Main Authors: Nievergelt, Adrian Pascal, Diener, Dennis Ray, Bogdanova, Aliona, Brown, Thomas, Pigino, Gaia
• Format: Article
• Language: English
• Subjects: Cell Biology; CRISPR; Genetics; Molecular Biology; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102774

CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line. We further empower this genetic-editing approach by efficient crossing and highly robust screening protocols. For complete details on the use and execution of this protocol, please refer to Nievergelt et al. (2023).1 [Display omitted] •CRISPR-Cas allows efficient gene disruption and endogenous protein tagging•Small-volume electroporation reduces reagent waste and improves cell viability•A highly robust (q)-PCR-based screening procedure reduces variability•FACS is used to increase the speed of crossing experiments Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line. We further empower this genetic-editing approach by efficient crossing and highly robust screening protocols.