CRISPR/Cas9 gene editing of Trypanosoma cruzi to study the functionality of the TcTASV multigene family

MASIP, Y; DE FREITAS NASCIMENTO, J; CHIDICHIMO, AM; COSENZA, M; SILBER, A; TEKIEL V

Buenos Aires

Congreso; XXXIII Reunion Anual Sociedad Argentina de Protozoología.; 2022

Sociedad Argentina de Protozoología

CRISPR/Cas9 gene editing of Trypanosoma cruzi to study the functionality of the TcTASV multigene familyTcTASV multigenic family is unique to Trypanosoma cruzi and is present in all the analyzed strains of the parasite. Its main subfamily, TcTASV-C, is located at the trypomastigote membrane and secreted both free and into extracellular vesicles, being expressed up to 100 times more in the blood circulating trypomastigotes than in those derived from in vitro cultures (Caeiro et al, 2018). TcTASV-C could have a role in the establishment of T. cruzi infection, although its exact function is still unknown.Here, we propose the use of the CRISPR/Cas9 technique to generate knock-out parasites for TcTASV-C (6 annotated genes in CL-Brener strain, TcVI). CRISPR/Cas9 requires the Cas9 nuclease, which generates a double-stranded cut in the region of interest guided by single guide RNAs (sgRNAs), being able to add a donor DNA for homologous recombination. We designed two sgRNAs -common for every TcTASV-C gene- and donor DNAs to replace TcTASV-C with blasticidin and/or puromycin resistance cassettes. This material was used to transfect CL-Brener epimastigotes constitutively expressing the Cas9 endonuclease and the T7 polymerase (Costa et al, 2018). We characterized clonal populations of blasticidin-resistant hemi knock-out parasites, analyzing genomic DNA by PCR with specific primers and subsequent sequencing. Thus, we determined that they have at least one wild-type gene copy (amplification with internal primers in TcTASV-C genes) and at least one interrupted copy (forward primer in the 5´UTR region of the gene and reverse primer on antibiotic resistance). No puromycin-resistant parasites were yet obtained. Blasticidin-resistant epimastigotes do not differ in morphology or growth rate from control. We have then obtained metacyclic trypomastigotes which infect Vero cells, differentiate into amastigotes and exit cells as trypomastigotes. Our next goal is to determine TcTASV-C mRNA (rt-qPCR) and protein levels in hemi knock-out trypomastigotes, to then proceed with the study of their in vivo and in vitro infective capacity.