Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis

ABSTRACT: Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C. reinhardtii. We have tested the strategy on two genes: LF5/CDKL5, lack of which causes a long-flagella phenotype, and Cre09.g416350/NAP1L1, which has not been studied previously in C. reinhardtii. We successfully tagged the C-terminus

SUBMITTER: Hou Y 

PROVIDER: S-EPMC9733891 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature