Development of Cas9 inducible linage of T. cruzi: a tool for genetic editing

• Grant number: 16/21283-9
• Support Opportunities: Scholarships abroad - Research
• Start date: May 01, 2017
• End date: October 31, 2017
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Simone Guedes Calderano
• Grantee: Simone Guedes Calderano
• Host Investigator: John Morrison Kelly
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Institution abroad: London School of Hygiene and Tropical Medicine, England

Abstract

CRISPR/Cas9 technology has been widely used as a very versatile and efficient method of genetic manipulation and is a very promising technique to be applied for studies in T. cruzi. Raising antibodies against specific proteins is time consuming and not always successful, the ability to tag a specific gene locus could facilitate the characterization of many proteins in this parasite. So far, the CRISPR/Cas9 system has been only used for gene knockdown in T. cruzi through a constitutive expression level of Cas9. However, Cas9 activity may interfere with some studies, for example, analysis of DNA replication and repair. I propose generating a cell line that can express Cas9 under tetracycline regulation using the pTcINDEX expression vector. This inducible Cas9 line would be able to provide high levels of Cas9 expression for a short period of time, which would be enough to allow the DNA editing but absent after that. Having such technology available in the lab would be a valuable working tool. (AU)

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)

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)