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

Abstract

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).

Keywords: CRISPR; Cell Biology; Microbiology; Model Organisms; Molecular Biology.

Graphical abstract

Figure 1

Representative scheme to design specific sgRNA molecules

Figure 2

Representative steps to knock out the target gene by CRISPR-Cas9 (A) Amplify the sgRNA scaffold. (B) Obtain sgTemplate DNA. (C) Generate the sgRNA by in vitro transcription. (D) Knock out target gene using the obtained sgRNA.

Figure 4

Location of tested sgRNAs and mutation of interest

Figure 5

Design of Cas9-cleavage resistant molecules

Figure 6

Obtention of DNA repair templates of the variants of the gene of interest by PCR amplification

Figure 3

Representative scheme of the construction of plasmids containing the DNA repair templates of the target gene

Figure 7

Cloning of the reconstitute gene variants to be used as templates in DNA recombination after Cas9 cleavage

Figure 8

Insertion of blasticidin-resistance cassette in ptcDAC2 plasmids containing the DNA repair templates of the variants of the gene of interest Note: ptcDAC2 plasmids are those obtained by cloning DAC2 gene variants (ptcDAC2_sens, ptcDAC2_resist and ptcDAC2_Y371F).

Figure 9

Generation of T. cruzi clones bearing the tcDAC2 DNA repair template plasmids for subsequent recombination after Cas9 cleavage

Figure 10

Complementation assay

Figure 11

Phenotypes observed in wild-type and tcDAC2-deficient T. cruzi cells (A and B) Deficient tcDAC2 cells present an abnormal phenotype with only one kinetoplast and multiple nuclei and flagella (A) suggesting proliferation defects and genome instability corroborated by the abnormal profile observed by cell cycle analysis (B). Cyan curves show wild-type cell profile and red and yellow line curves show mutant cell profiles. dpt – days post-transfection.