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Genetic manipulation of T. cruzi

Most of our current knowledge of the mechanisms controlling gene expression in trypanosomatids resulted from the development of transfection protocols, which allowed the manipulation of genes, the generation of knockout mutants and the introduction of reporter genes and genetic markers in parasite genomes. In contrast to T. brucei, in which homologous recombination of the foreign sequences with the parasite genome is the main strategy that allows the generation of stable transfection lineages, two types of transfection vectors are used in T. cruzi and in various Leishmania species. Vectors containing the foreign gene flanked by T. cruzi sequences allow the integration of the foreign DNA, by homologous recombination in the parasite genome. Episomal vectors have also been used to obtain high levels of expression of foreign genes, if they contain SL/polyadenyla- tion addition sites present both upstream (for trans-splicing) and downstream (for polyadenylation) from the exogenous gene (for a review, see Teixeira and daRocha69). Work from our lab has identified sequences derived from various genes that can be used to provide efficient frans-splicing and polyadenylation.70,71 With regards to the choice of promoters that can be used in T. cruzi expression vectors, we are quite limited. While VSG and procyclin promoters (both of them recognized by RNA polymerase I) work well in T. brucei expression vectors, the only option currently available in T. cruzi is the rRNA promoter. However, similar to what has been observed in various species of Leishmania, it is also possible to obtain relatively high levels of expression of foreign genes using episomal vectors that do not contain promoter sequences at all.46,72

An important breakthrough allowing a better control of genetic manipulation in trypanosomatids was achieved with the development of inducible expression of gene products under the control of tetracycline repressor. In this system, which has been initially developed for T. brucei, transgenic parasites expressing the tetracycline repressor of E. coli exhibit inducer (tetracycline)-dependent expression of a reporter gene cloned downstream from a trypanosome promoter bearing one or more copies of the Tet operator.73 Although such an inducible expression system has been developed in T. cruzi,71,74 its efficiency in controlling transcription in response to tetracycline does not seem to be as high as in T. brucei. More efforts are still needed to create better vectors to allow tight regulated, inducible expression of foreign genes in T. cruzi since the availability of such repressor/operator system is an excellent tool for dissecting function of essential genes and for expression of toxic gene products in the parasite.

A second major advance that provided a powerful tool for genetic manipulation in trypanosomes was described by Ngd et al.75 who were able to generate “knockdown” mutants by targeting mRNA degradation through the mechanisms of RNA interference (RNAi). RNAi is a very specific gene silencing mechanism guided by double strand RNA (dsRNA) bearing sequences derived from a target gene. Briefly, exogenously synthesized or internally expressed dsRNAs homologous to the coding sequence of a target gene are processed into 20—24—nt-long RNAs which work as active guides for mRNA degradation.76 RNAi is particularly convenient as a methodology to study trypanosomatid genes where conventional gene knockout is hindered by the fact that several genes are present in multiple copies.77 In addition to T. brucei, reports of successful RNAi knock-downs have been described for Trypanosoma congolense78 and in Leishmania braziliensis.79,80 Unfortunately, although RNAi has revolutionized genetic manipulation in T. brucei, the lack of an RNA silencing pathway in T. cruzi 71 and in old world Leishmania species79 has resulted in a much slower progress in similar studies in T. cruzi and in Leishmania.

Gene targeting by homologous recombination (HR) remains one of the most powerful techniques to investigate gene function since it allows the generation of parasites with defined mutations in their genome. If the target is not an essential gene, gene deletion by HR is initiated with the replacement of the first allele by a drug resistance marker and, in a second step, by transfecting the heterozygous mutant with a second resistance marker flanked by sequences corresponding to the second allele of the targeted gene. Although the first report of a T. cruzi gene knockout by HR was 20 years ago, this approach is time-consuming (3 months on average) and consequently, a limited number of studies involving generation of T. cruzi null mutants have been described.

More recently, new technologies developed to improve genetic manipulation in different organisms have been adapted for T. cruzi. Among the highly effective new tools that are now available to facilitate genome editing in T. cruzi are Cre- recombinases and the CRISPR-Cas9 system. Kangussu-Marcolino et al.81 showed that it is possible to create T. cruzi mutants using the conditional deletion DiCRE system by expressing the split CRE recombinase in epimastigotes. Following insertion of an expression cassette containing the gene encoding for puromycin-N-acetyl- transferase (purR) flanked by loxP sites in the T. cruzi genome, the report shows that induction of DiCRE recombinase by addition of rapamycin to the culture medium, resulted in the removal of the selectable marker with high efficiency. Soon thereafter, Peng et al.82 generated epimastigote cell lines stably expressing GFP and Cas9 nuclease and, after transfection with in vitro transcribed sgRNAs that target eGFP, these authors showed deletion of gfp sequences in 50—60% of parasites as early as day 2 after sgRNA transfection. Importantly, these authors also demonstrated that CRISPR-Cas9 system can be an effective in disrupting genes present in the parasite genome as multigene families such as a-tubulin and (3-galactofuranosyl glycosyl- transferase ((3-GalGT) genes. Subsequently, Lander et al.83 also reported the disruption of T. cruzi genes using CRISPR-Cas9 technology. These authors described two different transfection strategies, a single transfection vector carrying both the Cas9 nuclease and sgRNA sequences transcribed from the rRNA promoter and a strategy involving transfection of parasites with two separate plasmids, one containing Cas9 gene and the other carrying the sgRNA sequence, that allow generating knockout cell lines for genes encoding GP72 and paraflagellar rod proteins, which are protein required for flagellar attachment or components of the parasite flagellum. Certainly, entirely new avenues have been opened towards a more comprehensive functional analysis of this parasite’s genome.

 
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