Abstract
To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.
Keywords: CRISPR/Cas9, Endogenous tagging, Genome editing, Subcellular localization, Trypanosoma cruzi
Background
Genetic manipulation of protist parasites has significantly increased since the emergence of the CRISPR/Cas9 technology (Lander et al., 2016a). Trypanosoma cruzi is the causative agent of Chagas disease, which affects millions of people worldwide, particularly in Central and South America where the disease is endemic. Vaccines to prevent this disease have not been developed, and available drug treatments are not completely effective (Urbina and Docampo, 2003). This parasite has been particularly refractory to genetic manipulation (Docampo, 2011). However, the recent use of the CRISPR/Cas9 technology for gene knockout and knockdown (Peng et al., 2014; Lander et al., 2015) and to perform endogenous gene tagging (Lander et al., 2016b) has transformed the approaches for functional study of proteins in this organism. Here we describe a protocol to generate CRISPR/Cas9-mediated endogenous gene tagging in T. cruzi, leading to the expression of specific C-terminal tagged proteins in this parasite. Tagged proteins can be detected by Western blot analysis and their subcellular localization can be determined by immunofluorescence microscopy. Other potential applications of the technique include immunoprecipitation assays and tandem affinity purification (TAP) to establish protein-protein interactions.
Materials and Reagents
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Procedure
Data analysis
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Recipes
Acknowledgments
These experimental procedures have been published (Lander et al., 2016b). We acknowledge Mayara Bertolini for her assistance in recording Video 1. This work was funded by the São Paulo Research Foundation (FAPESP), Brazil (2013/50624-0) and the U.S. National Institutes of Health (grant AI107663). N.L. and M.A.C. are postdoctoral fellows of FAPESP (2014/08995-4 and 2014/13148-9).
References
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