GENERATING P21 GENE KNOCKOUT TRYPANOSOMA CRUZI BY CRISPR/CAS9 SYSTEM

TEIXEIRA, TL; PEREZ BRANDAN, C; CHIURILLO, MA; LANDER, N; BARROS, RRM; YONAMINE, CM; DASILVEIRA, JF; SILVA, CV

Caxambú

Congreso; Annual Meeting of the Brazilian XXXV Society of Protozoology. 46th Annual Meeting of Basic Reasearch in Chagas' disease; 2019

Sociedad Brasilera de Protozoología

In the process of interaction of Trypanosoma cruzi with the host cell, highly specific molecules of both organisms are involved. One of these molecules, P21, has been described as a protein involved in the invasion process of T. cruzi. More recent studies show that P21 is involved in the chronicity process ofChagas disease. Mice infected with T. cruzi and treated with P21 showed Increased collagen deposition and reduced angiogenesis in heart tissues damaged by the parasites. Also, P21 regulated intracellular replication in vivo and in vitro by modulating the parasite cell cycle. From this, what would be the effect of the absence of protein P21 on the parasite and in the course of infection? Thus, as a first step, this study aimed to produce P21 knockout parasites using the CRISPR/Cas9 technique. P21 is coded by a single copy gene located at chromosome TcChr22 of clone CL Brener. Two P21-specific guides were designedfrom different regions of P21 gene. Constructs containing the Cas9-EGFP gene and P21-specific single guides were produced and electroporated into epimastigotes of T. cruzi (Y strain). Transfected parasites were selected with G418 antibiotic, cloned by cell sorting, and kept in culture for 2 months. Followinggrowth of stable Cas9-single guide strains, parasites were electroporated with a donor DNA containing homology arms and a second selection antibiotic (blasticidin) marker. Antibiotic concentration was previously determined using WT parasites by viability and dose-response assays. Transfected parasiteswith P21-single guides showed extremely impaired growth when compared to scrambled control. Besides, after electroporation with donor DNA, more than 90% of parasites died in the presence of blasticidin after one week. Our data suggest that P21 plays key role in epimastigote replication or P21 is an essential gene that makes parasite survival impossible. Other approaches used CRISPR/Cas9 are being developed to elucidate our findings. Supported by:CNPq, CAPES, FAPESP.