CRISPR-CAS9 TARGET VALIDATION OF Trypano- soma cruzi BROMODOMAINS

PEZZA A; CRUZ BUSTOS T; ALONSO VL; TAVERNELLI LE; DOCAMPO R ; SERRA EC

Buenos Aires

Congreso; REUNION CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas ́ disease. This parasite has a complex life cycle with two intermediate hosts, a triatomine insect and a mammalian vertebrate. Nuclear gene organization in T. cruzi is atypical from other eukaryotic cells. Genes arrange in long tandems that are tran- scribed polycistronically from a small number of initiation sites. The transcription is driven by a limited number of basal nuclear factors and its regulation depends on chromatin structure. Among the usu- al proteins that participate in epigenetic regulation, a small number of genes coding for acetylases, deacetylases and bromodomains are present in the T. cruzi genome. Bromodomains are conserved protein modules capable of binding acetylated lysines and found in many proteins associated with chromatin. T. cruzi has at least six genes that encode for bromodomain-containing factors (TcBDF1-6). The overall objective of the project is to explore bromodomains as drug target in T. cruzi, by assaying their essentiality using a CRIS- PR/Cas9 gene deletion approach. Brie y, target sequences (SgR- NA) were cloned into the expression plasmid pTREX-Cas9-neo. Parasites were transfected by electroporation and drug selected. Deletion of the target genes were determined by PCR. We obtained mutated parasites for all the genes tested but only for Bdfx we got the homozygous population, which mean that this gene is not essen- tial for epimastigotes growth. For the rest of the genes we detected less amount of protein for mutated cultures but we couldn ́t get ho- mozygous null mutants, and only one of them showed a detrimental growth rate. We are evaluating the essentiality of these genes in the other life cycle stages as well as testing the expression of dom- inant-negative mutants approach for some of the genes. Altogether these results suggest that except Bdfx, bromodomains genes could be crucial for T. cruzi normal growth.