Life Sciences & Biotechnology
Title : | Repurposing the endogenous CRISPR-Cas system as an anti-Salmonella strategy |
Area of research : | Life Sciences & Biotechnology |
Focus area : | Microbiology, Genetic Engineering |
Principal Investigator : | Dr. Sandhya Amol Marathe, Birla Institute Of Technology And Science (BITS), Pilani, Rajasthan |
Timeline Start Year : | 2024 |
Timeline End Year : | 2027 |
Contact info : | sandhya.marathe@pilani.bits-pilani.ac.in |
Details
Executive Summary : | Salmonellosis, a foodborne disease, presents a formidable threat to humans. Salmonella has gained resistance against a wide range of antibiotics while the biofilms formed within the host and in the environment are recalcitrant to treatment. Biofilms formed within the host are responsible for recurrent infections and act as a reservoir for typhoid. In addition, there are no vaccines for non-typhoidal salmonellosis and typhoidal vaccines are not recommended for toddlers. This demands an effective and alternative therapeutic solution. Recently, exogenous CRISPR-antimicrobial has been explored as a sequence-specific alternative against Salmonella. However, there are limitations to this system owing to technicalities and the frequent emergence of escape mutants. The chief aim of the proposal is to harness the endogenous CRISPR-Cas3 system of Salmonella as an anti-microbial against itself. The endogenous CRISPR-Cas3 system offers an advantage over exogenous Cas9 as it significantly reduces the size of DNA to be transferred into the target pathogen and increases target specificity due to the requirement of larger crRNA (32bp for Cas3 versus 20bp for Cas9). As opposed to the frequent emergence of escape mutations with exogenous Cas9, the mutations in the endogenous CRISPR-Cas system are expected to be rare as it governs multiple physiological processes in Salmonella like biofilm formation and pathogenesis, and for serovar Typhi, Cas3 is also required for growth in LB media. Furthermore, the targeted DNA would have a lower repair probability as Cas3 shreds the DNA while the double-stranded DNA breaks created by Cas9 can be repaired. Thus, the killing efficiency with the endogenous system would be better. For utilizing endogenous CRISPR-Cas3 as an anti-microbial, the system would be activated using a transcriptional activator, LeuO, that would be supplied in trans. The functional activation would be assessed using plasmid loss assay in different Salmonella serovars as there exist two distinct cas operon types among the Salmonella serovars. The endogenous system would then be reprogrammed for self-genome degradation. For this, leuO and highly conserved Salmonella-specific spacers would be engineered as a CRISPR array on a conjugative plasmid. Non-pathogenic bacteria like lab strains of E. coli would be used to transfer this anti-Salmonella machinery into the bacteria using conjugation. Multiple protospacers on the core genome encoding essential virulence factors would be selected to minimize the emergence of mutants escaping self-targeting. Any mutations in these protospacers may render the virulence factor ineffective, thereby impacting the pathogenicity. This strategy might be an effective alternative therapy to exclusively eliminate Salmonella and its biofilms without harming the beneficial microorganisms. If successful, the scheme can be exercised on pathogens with endogenous CRISPR-Cas systems that are similar to that of Salmonella. |
Total Budget (INR): | 25,38,360 |
Organizations involved