Generation of T. cruzi (CL Brener) strains knockout for IP6K and evaluation of homologous recombination (HR) repair capacity

Abstract

In model eukaryotes, inositol pyrophosphates (PP-IPs) - mainly PP-IP4, IP7, and IP8 - are involved in a wide range of processes, such as regulation of telomere length, and homologous recombination (HR). However, the mechanism of action of PP-IPs in pathways related to DNA metabolism (especially homologous recombination repair) is not fully understood. PP-IP4, IP7, and IP8 are synthesized by pathways involving the participation of IP6K and PP-IP5K kinases. Trypanosomatids has an ortholog gene for IP6K, but apparently does not have orthologs for PP-IP5K, i.e., they do not synthesize IP8, which make them excellent models for the study of PP-IP4 e IP7. Thus, this MSc research plan consists of generating T. cruzi (CL Brener) strains KO to IP6K (IP6K-/+ e IP6K-/-) using the CRISPR/Cas9 system, challenge the strains generated by ionizing radiation (IR), and evaluate the HR repair capacity relative to WT using flow cytometry, growth curves, and protein recruitment kinetics (western blot - WB and immunofluorescence - IFA). Briefly, analysis of DNA content by flow cytometry will be done to identify possible cell cycle arrests and to measure the period required to overcome it. Growth curves will be performed to observe the proliferation pattern after IR, and comparative recruitment kinetics could identify subtle differences in the recruitment pattern of some key HR pathway components. It is worth emphasizing that this approach will be of fundamental importance to answer the essential questions raised by the main project. (AU)