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

• Published in: STAR protocols Vol. 3; no. 2; p. 101324
• Main Authors: Picchi-Constante, Gisele Fernanda Assine, Hiraiwa, Priscila Mazzocchi, Marek, Martin, Rogerio, Vanessa Zulkievicz, Guerra-Slompo, Eloise Pavão, Romier, Christophe, Zanchin, Nilson Ivo Tonin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-06-2022; Elsevier
• Subjects: Cell Biology; Chagas Disease - genetics; CRISPR; CRISPR-Cas Systems - genetics; Gene Editing - methods; Gene Knockout Techniques; Genes, Essential; Genetics; Humans; Life Sciences; Microbiology; Model Organisms; Molecular Biology; Protocol; Trypanosoma cruzi - genetics; CRISPR; Model Organisms; Microbiology; Molecular Biology; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101324

This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, 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 T. cruzi.
For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).
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•Genetic complementation strategy to investigate gene function in Trypanosoma cruzi•Analysis of specific mutations on protein function in vivo•CRISPR-Cas9-based gene knockout and gene replacement•Rapid analysis of Trypanosoma cruzi essential genes
Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, 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 T. cruzi.