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

MASIP, YAMIL E.; DE FREITAS NASCIMENTO, JANAINA; CHIDICHIMO, AGUSTINA; COSENZA, MAXIMILIANO; SILBER, ARIEL; TEKIEL, VALERIA

Buenos Aires

Congreso; XXXIII Reunión Anual SAP 2022; 2022

TcTASV multigenic family is unique to Trypanosoma cruzi and is present in all theanalyzed strains of the parasite. Its main subfamily, TcTASV-C, is located at thetrypomastigote membrane and secreted both free and into extracellular vesicles, beingexpressed up to 100 times more in the blood circulating trypomastigotes than in thosederived from in vitro cultures (Caeiro et al, 2018). TcTASV-C could have a role in theestablishment of T. cruzi infection, although its exact function is still unknown.Here, we propose the use of the CRISPR/Cas9 technique to generate knock-outparasites for TcTASV-C (6 annotated genes in CL-Brener strain, TcVI). CRISPR/Cas9requires the Cas9 nuclease, which generates a double-stranded cut in the region ofinterest guided by single guide RNAs (sgRNAs), being able to add a donor DNA forhomologous recombination. We designed two sgRNAs -common for every TcTASV-Cgene- and donor DNAs to replace TcTASV-C with blasticidin and/or puromycinresistance cassettes. This material was designed and used to transfect CL-Brenerepimastigotes constitutively expressing the Cas9 endonuclease and the T7polymerase (Costa et al, 2018). We characterized clonal populations of blasticidin-resistant hemi knock-out parasites, analyzing genomic DNA by PCR with specificprimers and subsequent sequencing. Thus, we determined that they have at least onewild-type gene copy (amplification with internal primers in TcTASV-C genes) and atleast one interrupted copy (forward primer in the 5'UTR region of the gene and reverseprimer on antibiotic resistance). No puromycin-resistant parasites were yet obtained.Blasticidin-resistant epimastigotes do not differ in morphology or growth rate fromcontrol. We have then obtained metacyclic trypomastigotes which infect Vero cells,differentiate into amastigotes and exit cells as trypomastigotes. Our next goal is todetermine TcTASV-C mRNA (rt-qPCR) and protein levels in hemi knock-outtrypomastigotes, to then proceed with the study of their in vivo and in vitro infectivecapacity.