Department of Chemistry and Biochemistry - University of Oklahoma

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Host: Dipali Sashital

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“Mechanisms of CRISPR-Cas Systems”

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection against intruding genomic materials in bacteria and archaea. The complementarity of CRISPR RNA (crRNA) guides Cas protein to a target DNA and/or RNA, which then initiates strand cleavage by a specific Cas protein. Cas9, a type II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA (sgRNA) can direct Cas9 to cleave DNA targets of interest.

In addition to the complementarity between the guide region of the sgRNA and the target DNA, a three to eight nucleotide long region, termed protospacer adjacent motif (PAM), is essential for Cas9 function. The relationship between RNA-mediated conformational changes in Cas9 and DNA targeting is being pursued actively for developing Cas9 variants with high specificity for gene editing. Our lab has focused our efforts on identifying how an arginine-rich bridge helix (BH) present in Cas9 contributes to conformational checkpoints essential for stringent DNA cleavage (1). Cas9 offers great promises for gene therapy applications and one critical aspect for such applications is to completely understand the DNA cleavage requirements of Cas9 and identify any promiscuous DNA cleavage by this protein.

Interestingly, our research has identified conditions where Cas9 and other Cas proteins can cleave DNA in the absence of a guide RNA, under in vitro conditions (2).