Biophysics Colloquium by David Taylor (University of Texas - Austin)

David Taylor
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.

Hosted by: Elizabeth Kellogg

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