CREditing: a tool for gene tuning in Trypanosoma cruzi

Abstract

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Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi

“…For T. cruzi, successful editing can be achieved by different methods such as the stable expression of SpCas9 followed by the transient transfection with in-vitro-transcribed sgRNA (Peng et al, 2014;Burle-Caldas et al, 2018;Romagnoli et al, 2018), the stable expression of both SpCas9 and sgRNA (Lander et al, 2015), the stable expression of T7 RNA polymerase and SpCas9 followed by the transient transfection of a DNA template for the sgRNA expression directed by the T7 promoter (Costa et al, 2018), and last but not least, the marker free editing by the transfection of the ribonucleoprotein complex (SaCas9 + sgRNA) protein SaCas9 (Soares Medeiros et al, 2017;Burle-Caldas et al, 2018). Recombinant protein electroporation was also applied for CRE recombinase delivery to manipulate T. cruzi gene expression (Pacheco-Lugo et al, 2020). By applying the SaCas9 protein delivery, we easily disrupted TcTrypanin using a donor oligonucleotide to insert stop codons plus a restriction site, when compared with the conventional approach.…”

The long and winding road of reverse genetics in Trypanosoma cruzi

“…Although the system is not inducible due to the apparent endogenous production of glucosamine 6-phosphate, it has resulted useful to obtain knockdowns of essential genes in T. cruzi [ 35 , 36 ]. In addition, the CRE- lox recombination system has been tested in T. cruzi epimastigotes and tissue culture trypomastigotes [ 37 ]. Albeit it has not been used to manipulate endogenous genes yet, the adaptation of the CRE- lox system to generate an inducible CRISPR/Cas9 knockout strategy could contribute to expand the available toolbox to manipulate this parasite.…”