Construction of transgenic lines modified by CRISPR/Cas9 to identify events of aneuploidy in Trypanosoma cruzi

Abstract

Genomic analysis indicates that T. cruzi is diploid for most of its chromosomes. However, aneuploidy events were detected in some chromosomes by aCGH and NGS (next generation sequencing). It has been suggested that aneuploidy may be an important factor in generating genetic diversity in T. cruzi, an organism in which sexual reproduction is absent or very rare. By comparing strains and clones of T. cruzi with aCGH, we identified deletion or duplication events in small chromosome segments (segmental aneuploidy). We also identify changes throughout the length of the chromosome, with duplication or loss of a complete chromosome. In Leishmania, there are subpopulations with different degrees of ploidy in one cell population for one or more chromosomes, resulting in a structure known as "mosaic aneuploidy". The variation of ploidy has been related to resistance to chemotherapeutics in clinical isolates of L. donovani. Aneuploidy is an emerging phenomenon in T. cruzi with several open questions, such as: What are the mechanisms responsible for aneuploidy in T. cruzi? Is there mosaic aneuploidy and what would be the role of this phenomenon in this parasite? We intend to investigate the presence of aneupoidia in strains of T. cruzi as well as in clones derived from the same parental strain. The FISH technique is the most appropriate because it allows to estimate the number of aneuploid cells in the population and to identify the chromosomes involved in the process. However, due to the small size of the T. cruzi nucleus and chromosomes, the FISH technique has to be improvedto detect single copy sequences. In this project we propose a new strategy to identify specific target sequences of the TcChr24 chromosome of the CL strain and the CL Brener clone (CLB) of T. cruzi by inserting tags using CRISPR / Cas9. To achieve this goal, the following steps are planned: 1) To identify chromosome-specific sequences (target sequences for FISH) on the TcChr24 chromosome (T. cruzi aneuploidy); 2) To generate transgenic CL and CLB clone lines with TcChr24 chromosome, replacing the target sequences for FISH by the blasticidin gene in fusion with exogenous repeats without similarity in the T. cruzi genome by the CRISP/Cas9 technique; 3) To analyze events of aneuploidy throughout the generations in the CL strain and CLB clone.