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We developed a strategy to identify the endogenous gene in situ to avoid 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. In C. reinhardtii, the tactic uses TIM, a CRISPR/Cas9-based technique for targeted insertional mutagenesis. Cre9. g416350/NAP1L1, which has never been studied before in C. reinhardtii, has been tested on two genes: LF5/CDKL5, a lack of which causes a long-flagella phenotype, and Cre9. g416350/NAP1L1. We successfully tagged the C-terminus of wild-type LF5 with the hemagglutinin tag, which had a yield of 7. 4%. This is the first time that C. reinhardtii endogenous genes have been modified in situ to produce a wild-type tagged protein. In C. reinhardtii, this effective, cost-effective, and convenient TIM-tagging scheme promises to be a useful instrument for the study of nuclear genes, including essential genes.
Source link: https://europepmc.org/article/MED/36490276
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