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Development of an inducible RNA-targeting CRISPR-Cas system for Trypanosoma cruzi

Grant number: 19/23180-0
Support type:Scholarships abroad - Research Internship - Post-doctor
Effective date (Start): May 01, 2020
Effective date (End): October 31, 2020
Field of knowledge:Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
Principal Investigator:Ariel Mariano Silber
Grantee:Janaina de Freitas Nascimento
Supervisor abroad: John Morrison Kelly
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Local de pesquisa : London School of Hygiene and Tropical Medicine, England  
Associated to the scholarship:17/12320-0 - Characterization and biological role of histidine catabolism in Trypanosoma cruzi, BP.PD

Abstract

Over the past 30 years, gene function studies in Trypanosomatids have employed tools such as heterologous expression, gene overexpression and knock out. The recent adaptation of the bacterial CRISPR-Cas system as a molecular tool expanded the toolbox for genetic manipulation in these organisms. However, so far, all the CRISPR-Cas systems that have been modified to allow gene expression manipulation in Trypanosoma cruzi target DNA. There have been two major drawbacks: these systems either require selection of the mutant parasites with antibiotics or extensive screening to find the parasites with the correct mutation. To date, there is no available inducible and regulatable system to induce gene expression knockdown in T. cruzi. It makes impossible to study genes essential to parasite viability and which complete gene knockout would not be tolerated by the cell. Here we propose the development of an inducible RNA-targeting CRISPR-Cas system for T. cruzi to allow RNA knockdown based on bacterial CRISPR-Cas9 and Cas13 which have RNA cleavage activity. As an initial target, we propose the first enzyme of the His degradation pathway, the histidine ammonia lyase - TcHAL, since it is not essential for parasite viability. This system would facilitate the investigation of the phenotypes associated with the reduction of the expression of the enzymes involved in biochemical reactions crucial to T. cruzi's survival. Beyond gene expression knockdown, the uses for an inducible RNA-targeting CRISPR-Cas system would include facilitating genome-wide high-throughput loss- and gain-of-function screens, which would have many applications, including drug mode of action studies. (AU)