Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi

Abstract

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.

Transcription activity contributes to the firing of non-constitutive origins in Trypanosoma brucei

“…Such a proposal has previously been supported in this parasite after the induction of DSBs by ionizing radiation (IR), where repair was carried out quickly in late S/G2 within approximately 6 h (39). Thus, we can infer that endogenous DNA damage does not appear to be a great obstacle for this parasite to continue to proliferate, given the efficient DNA repair that most trypanosomatids possess (39,52,59,60).…”

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

“…(i) The method involves the use of a parental cell line expressing Cas9 and T7 RNA polymerase (T7 RNA Pol) for nucleofection of three PCR products: two donor DNAs with different resistance markers (RM 1 and RM 2) and a DNA fragment holding the specific sgRNA sequence downstream the T7 promoter for in vivo transcription; (ii) In the nucleus of the cell transiently expressed sgRNA assembles with Cas9; (iii) Cas9 targets both alleles of the gene, which are simultaneously replaced by two different resistance markers. more efficient than MMEJ in trypanosomatids Burle-Caldas et al 2018;Chiurillo et al 2016;Lander et al 2015;Sollelis et al 2015;Vasquez et al 2018;Zhang and Matlashewski 2015). Other factors could affect the efficiency of the system, such as the DNA delivery system (nucleofection is a more efficient electroporation method than transfection); the possibility of performing cell sorting during the selection period, to enrich the population of genetically modified parasites or even get mutant clones; and the presence of a resistance marker in the DNA donor template to ensure the selection of parasites with properly edited genomes.…”

“…The main advantage of this method is the possibility of transfecting different sgRNAs simultaneously, which could be useful for targeting several genes. However, in the absence of a DNA donor template to induce HDR, the double-strand break produced by Cas9 would be repair by MMEJ, which is a less efficient DNA repair mechanism in trypanosomatids Burle-Caldas et al 2018;Chiurillo et al 2016;Lander et al 2015;Sollelis et al 2015;Vasquez et al 2018;Zhang and Matlashewski 2015). In addition, the transient expression of in vitro-transcribed sgRNAs could reduce the efficiency of the method.…”

An Evolutionary View of Trypanosoma Cruzi Telomeres

“…In other words, the pTACs showed nondetectable sequence exchange with the host chromosomes and replicated and segregated without the presence of centromeres, or perhaps the telomere was fulfilling this role. In a more recent experiment (Chiurillo et al, 2016b), addressed the possibility of chromosomal sequence exchanges by introducing a cutting site for the rare meganuclease I-SceI within the RHS gene of one pTACs (pTAC-D6CISceI * ) harboring larger portions of T. cruzi subtelomeres. After transforming this pTAC into T. cruzi cells expressing the meganuclease I-SceI, and confirming that double-strand breaks (DBSs) were produced, the probing pTAC was examined to check whether the DSBs were repaired.…”

“…Also, they are found inserted downstream the sequence GA (x) 2 AxGa (x) 5 txTATG↑A(x) 11 ↑ where arrows mark the single strand cleavage sites (El-Sayed et al, 2005;Bringaud et al, 2006). How frequent DBSs leading to genetic recombination and gene variability occurs is difficult to assess, since as shown in the experiments with meganuclease I-Sce-I (Chiurillo et al, 2016b), DBSs are mainly repaired by HR using as template homolog chromosomes.…”