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Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in

STAR Protoc. 2022-04; 
Gisele Fernanda Assine Picchi-Constante, Priscila Mazzocchi Hiraiwa, Martin Marek, Vanessa Zulkievicz Rogerio, Eloise Pavão Guerra-Slompo, Christophe Romier, Nilson Ivo Tonin Zanchin
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Plasmid DNA Preparation … Mix 100 μL of cells and 20 μg of Cas9/pTREX-n plasmid in the test tube. … Note: In this work, the sgRNA scaffold sequence was acquired cloned into pUC19 from GenScript, but the … Get A Quote

摘要

This protocol outlines a new genetic complementation strategy to investigate gene function in , the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in . For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).

关键词

CRISPR, Cell Biology, Microbiology, Model Organisms, Molecular Biology