Rajan, RakhiMartin, Lindsie2024-07-292024-07-292024-08-01https://hdl.handle.net/11244/340523CRISPR-Cas systems provide adaptive immunity to bacteria and archaea. They do this by a CRISPR RNA (crRNA)-led Cas nuclease. The Cas protein recognizes the invader genome based on signature motifs present only in the invader [for example a 2-8 nucleotides long DNA motif called the protospacer adjacent motif (PAM)], followed by complementary base pairing between crRNA and the invader genome forming an “R-loop”. The R-loop formation triggers the nuclease activity of the Cas protein, effectively neutralizing the invasion. Recently mechanisms of Cas9 and Cas12a nucleases have been used to develop gene therapies and molecular diagnostics tools. Cas9 is the most-used gene editing tool at present, but Cas12a has favorable features for in vivo gene editing because of its smaller size, crRNA processing ability, and creation of staggered double-stranded DNA (dsDNA) cleavage post editing that enhances recombination events. Gene editing with these Cas proteins, however, has some setbacks. Even though complementarity between the crRNA and target DNA is necessary for dsDNA cleavage to occur, some mismatches between them are tolerated and lead to off-target cleavage. Cas9 and Cas12a have also been shown to cleave or degrade DNA non-specifically in the absence of a crRNA (RNA-independent, R-I) or after being activated of crRNA-dependent DNA cleavage (called as trans activity). All these non-specific activities (off-target, RNA-independent, and trans cleavages) can cause unwarranted genome changes following the use of Cas proteins in genome applications. To increase its safety and to promote it as a mainstream gene editing tool, we focused on removing off-target cleavage of Cas12a and screen for decreases in non-specific activities such as R-I and trans DNA cleavage activities. We used protein engineering of a conserved arginine-rich “bridge helix” of Cas12a that was shown to play an integral role in mediating conformational changes of Cas12a needed for DNA cleavage and to impart specificity in DNA cleavage. We also assess the role of Cas9 protein in alternate functions such as promoting bacterial virulence. We used a bioinformatics-based protein sequence analysis to identify motif(s) in the Cas9 sequence that are uniquely present only in pathogenic bacteria. These studies combined will enhance our knowledge of CRISPR-Cas systems that will lead to safer gene editing tools and expand the CRISPR toolbox to include novel antibiotic targets.CRISPRCas12aCas9