Multiplex CRISPR/Cas9 gene editing - generation of unmarked knockout BCG compatible with DIVA skin test

• Grant number: 21/13879-7
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Effective date (Start): July 01, 2022
• Effective date (End): June 30, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Luciana Cezar de Cerqueira Leite
• Grantee: Luana Moraes
• Supervisor: Johnjoe McFadden
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Research place: University of Surrey, England
• Associated to the scholarship: 17/17218-0 - Implantation of the CRISPR-Cas9 platform in mycobacteria: investigation of ESX-1 system in the immunogenicity of BCG, BP.DD

Abstract

Tuberculosis (TB) is a global health burden. Besides the public health issue, the disease is also an economic problem in livestock production. Vaccination is the most cost effective strategy for TB control. So far, the only licensed vaccine is BCG, a live attenuated strain of Mycobacterium bovis. Although it carries several advantages, BCG protection declines over time, ranging from 0-80% in adults. The best scenario to control the disease is the combination of vaccination and early diagnostic. In bovine, the control of TB is based on test-and-cull, which reinforce the importance of a more accurate diagnostic. The tuberculin skin test (PPD) is currently used, but it does not distinguish BCG from other mycobacteria (pathogenic and environmental). A new diagnostic test capable of Differentiating Infected from Vaccinated Animals (DIVA) has been developed, based on immunodominant antigens present in M. bovis and absent in BCG; inclusion of additional antigens shown dispensable to in vivo persistence of the bacillus has displayed increased sensitivity. A DIVA-compatible BCG KO deficient of these antigens has been constructed through a very laborious and time consuming prosses; elimination of the antibiotic resistance genes from the genome will be required. We have shown the efficiency and versatility of CRISPR/Cas9 for the generation of unmarked KOs. We propose to induce multiple gene knockouts to generate an unmarked DIVA-compatible BCG KO strain. This strain will be transformed to obtain expression of the adjuvant LTAK63 to improve protection against MTB/BTB challenge. (AU)