Genetic edition and drug free selection in Trypanosoma cruzi: new methodology for modifications by CRISPR/Cas9

Abstract

Chagas disease is caused by Trypanosoma cruzi, an important endemic disease in tropical countries which has been considered a neglected disease by the World Health Organization (WHO). Thus, the study and understanding of this parasite are fundamental to develop new and better treatments. Gene editing by CRISPR/Cas9 together with drug resistance gene insertion has been widely used as a method of positive selection of recombinant T. cruzi. However this method has a few inconvenient such as antibiotic high cost; multiple genes tag difficulty, primarily due to low variety of available antibiotics; change of the original UTR of the target genes, which is important for stabilization and translational modulation of the mRNAs in this parasite. Regarding these difficulties, this project aims to validate a new methodology where the addition of tag sequences on targeted genes will be done without the addition of drug resistance genes. So, clonal selection of recombinant parasites will be used to obtain a monoclonal population. Utilizing CRISPR/Cas9 tool as well as PCR products, tag sequences can be inserted on the 5' or 3' portion of the targeted gene, causing little modification on the original UTRs. Afterward of the transfection and cloning process, the positive populations will be identified and the gene modifications and protein expression assessed. It will also be assessed the possibility of simultaneous modification of different genes utilizing the technique proposed.