Studies on the endogenous CRISPR/Cas system of BCG and its application as a biotechnological tool

• Grant number: 24/22895-4
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: June 01, 2025
• End date: March 31, 2030
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Luciana Cezar de Cerqueira Leite
• Grantee: Izadora Dillis Faccin
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 17/24832-6 - Development of vaccines based on recombinant BCG: Tuberculosis, Pertussis, Pneumococcus and Schistosoma, AP.TEM

Abstract

Bacillus Calmette and Guérin (BCG) is a widely used live attenuated vaccine against tuberculosis (TB). However, its efficacy in preventing pulmonary TB in adults is limited and variable. Gene editing in BCG has enabled advances in the study of immune response evasion mechanisms and in the development of new vaccines. The CRISPR/Cas9 tool, with its great biotechnological potential, allows the introduction or removal of genomic sequences using the guide RNA-directed Cas9 endonuclease and the host organism's DNA repair systems. Despite its potential, the application of CRISPR/Cas9 in BCG faces challenges related to efficiency and toxicity. Alternatively, the endogenous CRISPR system present in mycobacteria offers a promising approach, eliminating the need for heterologous expression of Cas9 and allowing the generation of multiple guide RNAs for the simultaneous editing of several genes. This project proposes to explore the endogenous CRISPR system of BCG, focusing on its biotechnological application. The objectives include investigating the gene expression profile of components related to DNA repair mechanisms and the endogenous CRISPR system in mycobacteria; constructing mycobacterial vectors to introduce mutations in genes that facilitate phenotypic screening; and optimizing conditions for the generation of knock-out and knock-in mutations. The project aims to develop a robust tool for gene editing in BCG, contributing to new opportunities for genetic engineering and the development of vaccines based on this bacillus. (AU)