APPLICATION OF CRISPR/Cas9 in BCG: Comparative assessment of Cas9 orthologs in generating genetic knockouts

• Grant number: 23/05432-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2023
• End date: July 31, 2024
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Alex Issamu Kanno
• Grantee: Beatriz Squinca D'Anníbale
• Host Institution: Instituto Butantan. São Paulo , SP, Brazil

Abstract

The BCG vaccine, the only one available to prevent tuberculosis, was developed at the beginning of the 20th century by Albert Calmette and Camille Guérin. BCG is a live-attenuated vaccine generated through successive cultures of the pathogenic Mycobacterium bovis. It prevents tuberculosis in a safe and effective way in children, however, the protection guaranteed by the vaccine in teenagers and adults is variable, exposing these individuals to pulmonary tuberculosis: the deadliest form of the disease. In this context, new and improved vaccines based on BCG are under development, even with methodological barriers restraining the manipulation of the BCG genome. Two of the most promising vaccines currently in advanced clinical trials required technically challenging steps in order to successfully modify the vaccine's genome and generate the vaccine candidate. This project aims to exploit CRISPR/Cas9 as a molecular tool to easily manipulate mycobacterial genomes. Previous data from our lab demonstrate the feasibility of CRISPR/Cas9 to generate knock-outs in BCG, although it also identified several challenges that need to be overcome. This project aims to study an alternative to the original Streptococcus pyogenes Cas9 and evaluate different scenarios for its optimized activity in BCG. If successful, improved CRISPR/Cas in BCG would enable several other studies to take place which require genome manipulation and may move one step forward in the development of new and improved tuberculosis vaccines.