title: Editing the Trypanosoma cruzi genome with zinc finger nucleases.
abstract: Gene function studies in Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, have been hindered by the lack of efficient genetic manipulation protocols. In most organisms, insertion and deletion of DNA fragments in the genome are dependent on the generation of double-stranded DNA break (DSB) and repair. By inducing a site-specific DSB, zinc finger nucleases (ZFNs) have proven to be useful to enhance gene editing in many cell types. Using a pair of ZFNs targeted to the T. cruzi gp72 gene, we were able to generate gp72 knockout parasites with improved efficiency compared to the conventional gene knockout protocol. We also provide evidence that, in T. cruzi, repair of DSBs generated by ZFNs occurs primarily by the homologous recombination pathway.
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors.forename: Gabriela Assis
authors.initials: GA
authors.lastname: Burle-Caldas
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors.forename: Viviane
authors.initials: V
authors.lastname: Grazielle-Silva
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors.forename: Melissa
authors.initials: M
authors.lastname: Soares-Simões
authors.affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Schumann Burkard
authors.affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.
authors.forename: Isabel
authors.initials: I
authors.lastname: Roditi
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Brazil.
authors.forename: Wanderson Duarte
authors.initials: WD
authors.lastname: DaRocha
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Electronic address: santuzat@ufmg.br.
authors.forename: Santuza M
authors.initials: SM
authors.lastname: Teixeira
mesh_terms: D004720:Endonucleases / Q000378:metabolism*; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D018503:Genome, Protozoan; D023281:Genomics / Q000379:methods; D014349:Trypanosoma cruzi / Q000235:genetics*; D016335:Zinc Fingers
chemical_list: D004720:Endonucleases
keywords: Gene knockout; Genome editing; Trypanosoma cruzi; Zinc finger nucleases; gp72