STRUCTURAL AND FUNCTIONAL INSIGHTS INTO CRISPR-CAS9 CATALYTIC ACTIVATION AND SPECIFICITY ENHANCEMENT
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CRISPR/Cas9 has been repurposed as a powerful genome editing tool, with immense potential toward therapeutic applications. Despite recent advances in understanding DNA recognition and cleavage by Cas9, consistent structural and functional information about the catalytic state of the HNH nuclease domain remains to be elucidated. On the basis of our recent work (Nature Scientific Reports, 7:17271), we here report a new Cas9 active state complex structure discovered by molecular dynamics simulations and validated by site-directed mutagenesis experiments. In this structure, the HNH domain is poised for cleaving the target DNA strand using a canonical catalytic triad as seen in phage T4 Endonuclease VII. Guided by the derived new structural information, we rationally designed and tested a library of new Cas9 variants to improve Cas9 specificities. Our ultimate goal is to offer several high-fidelity Cas9 enzymes that can be broadly used in basic research and medical therapy.