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Wednesday, February 8, 2023 at 4:00pm
Clark Hall, 701 Central Campus, 142 Sciences Drive
Associate Professor of Molecular Biosciences, University of Texas at Austin
Reengineering CRISPR-Cas effector complexes
CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.
Speaker Biography: David Taylor is an Associate Professor in the Department of Molecular Biosciences. He started as an Assistant Professor at UT Austin in September 2016. He earned his Ph.D. at Yale University and performed postdoctoral training at UC Berkeley with Eva Nogales and Jennifer Doudna. He has a passion for RNPs. He loves the Warriors, Syracuse basketball, teaching, and ‘starting’ projects that other lab members actually finish.
Hosted by: Elizabeth Kellogg
~ Coffee and tea provided ~
Open to the public
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