Thursday, November 21, 2024
11/21/2024
Project Summary/Abstract Bacteria employ several distinct defense strategies for protection from phage infection. CRISPR-Cas is one such mechanism that provides an adaptive immune protection against recurring phage infections based on genetic memory retained from past infections. The genetic memory is retained through a process called “adaptation” where short pieces of the intruder DNA (called prespacers) are selected following certain rules, and site-specifically inserted into the CRISPR locus as a “spacer”. The spacer is transcribed into guide-RNAs that are essential for sequence-specific targeting and inactivation of foreign genetic material. Adaptation in type II- A CRISPR systems is unique compared to other CRISPR systems due to the inherent capabilities of Cas1 and Cas2 proteins to catalyze the site-specific prespacer insertion without assistance from cellular factors to maintain fidelity during insertion. This property offers unique promises for type II-A Cas1-Cas2-based biotechnological and biomedical applications. Previous work from the PI’s group has identified distinct subgroups of type II-A CRISPR systems based on conserved DNA motifs present at the site of insertion, which is conserved across many bacterial genera. Accompanying work had biochemically established unique differences in the mechanisms of prespacer insertion by the different subgroups. The proposed research aims to derive molecular mechanisms of the differences in DNA requirements and efficiencies of prespacer insertion between the different type II-A subgroups. To derive structural and conformational differences between these subgroups, hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations will be performed, followed by biochemical validation of the results from HDX-MS and MD simulations. To assess the contribution of individual protein and nucleic acid complexes in site-specific DNA insertion, QM/MM (quantum mechanics/molecular mechanics) calculations will be employed. Based on the free energy contributions identified, amino acids and DNA nucleotides will be mutated to change integration efficiency with the end goal of engineering new Cas1-Cas2 variants which has developed specificity to a newly designed DNA sequence. Since Cas1 and Cas2 based prespacer insertion is vital to the functioning of all CRISPR systems, the lessons learned can be translated to other CRISPR systems. Applications include programmed DNA insertions, enhancing phage therapy to fight drug-resistant bacteria, and to develop bacteria that can fight off phage infections for the fermentation industry.
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