Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants

• Published in: International journal for parasitology 2018-07, Vol.48 (8), p.591-596
• Main Authors: Burle-Caldas, Gabriela Assis, Soares-Simões, Melissa, Lemos-Pechnicki, Laiane, DaRocha, Wanderson Duarte, Teixeira, Santuza M.R.
• Format: Article
• Language: English
• Subjects: CRISPR/Cas9; Genome editing; GP72; Recombinant Staphylococcus aureus Cas9; RNP complex; Trypanosoma cruzi
• ISSN: 0020-7519; 1879-0135
• DOI: 10.1016/j.ijpara.2018.02.002

[Display omitted] •We obtained fast generation of Trypanosoma cruzi GP72 knockout using Cas9 and in vitro transcribed single guide RNA.•Trypanosoma cruzi knockout mutants were generated using recombinant Cas9.•Highly efficient CRISPR/Cas9 editing protocols were used for T. cruzi without drug selection. CRISPR/Cas9 technology has been used to edit genomes in a variety of organisms. Using the GP72 gene as a target sequence, we tested two distinct approaches to generate Trypanosoma cruzi knockout mutants using the Cas9 nuclease and in vitro transcribed single guide RNA. Highly efficient rates of disruption of GP72 were achieved either by transfecting parasites stably expressing Streptococcus pyogenes Cas9 with single guide RNA or by transfecting wild type parasites with recombinant Staphylococcus aureus Cas9 previously associated with single guide RNA. In both protocols, we used single-stranded oligonucleotides as a repair template for homologous recombination and insertion of stop codons in the target gene.