Exploring the use of new genome editing technology as a fast-track approach to studying topoisomerase biology in Trypanosoma cruzi

• Grant number: 16/08958-7
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: September 06, 2016
• End date: September 05, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Glaucius Oliva
• Grantee: Fernanda Cristina Costa
• Supervisor: John Morrison Kelly
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Institution abroad: London School of Hygiene and Tropical Medicine, England
• Associated to the scholarship: 14/15145-7 - Structural studies of mitochondrial topoisomerase II from trypanosomatids, BP.PD

Abstract

The protozoan parasite Trypanosoma cruzi is an important zoonotic pathogen which causes Chagas disease. The genetic complexity of T. cruzi, as well as the limited set of efficient techniques for genome engineering, have contributed to the lack of progress in understanding and studying this pathogen. The CRISPR-Cas9 system has been adapted as a powerful tool for genome manipulation in T. cruzi. It has enabled the study of essential genes and their functions, and facilitated the analysis of large multi-gene families. However, Cas9 can cleave off-target sites, which poses a major challenge for genome editing. Recent studies have attempted to genetically engineer Cas9 enzymes to reduce off-target effects, to maintain robust on-target cleavage, and to provide useful tools for genome editing with high level specificity. We propose here to assess the potential of these newly modified Cas9 enzymes in T. cruzi, focussing on topoisomerase genes as a proof of principle. Trypanosomatids contain 6 topoisomerase genes. These have only been studied in detail in T. brucei, mainly because of the availability of RNAi machinery in this organism. Topoisomerase inhibitors are amongst the most effective and most commonly used anticancer and antibacterial drugs, so understanding the biology of each topoisomerase in T. cruzi would shed light on new molecular targets with potential for chemotherapy. Systematic disruption of the topoisomerase genes using newly available Cas9 enzymes, should lead to a better understanding of the role(s) of topoisomerases, as well as optimising the application of CRISPR-Cas9 technology to T. cruzi.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

OLMO, FRANCISCO; COSTA, FERNANDA C.; MANN, GURDIP SINGH; TAYLOR, MARTIN C.; KELLY, JOHN M.. Optimising genetic transformation of Trypanosoma cruzi using hydroxyurea-induced cell-cycle synchronisation. Molecular and Biochemical Parasitology, v. 226, p. 34-36, DEC 2018. (13/07600-3, 16/08958-7)

COSTA, FERNANDA CRISTINA; FRANCISCO, AMANDA FORTES; JAYAWARDHANA, SHIROMANI; CALDERANO, SIMONE GUEDES; LEWIS, MICHAEL D.; OLMO, FRANCISCO; BENEKE, TOM; GLUENZ, EVA; SUNTER, JACK; DEAN, SAMUEL; et al. Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. PLoS Neglected Tropical Diseases, v. 12, n. 4, APR 2018. (16/21283-9, 13/07600-3, 16/08958-7)