Improving Speed and Fidelity of CRISPR/Cas9: Kenneth Johnson, PhD, and David Taylor, PhD

“Since this region's not important, we think, for on-target activity, changing the activity on the off-target is fine. Its only role is apparently in helping to find this mismatch and saying it's okay to be cleaved there. So, by distorting the loop, we're taking that signal away so that it will recognize that it is a mismatch. But we're also not interfering at all with how the enzyme would work on a perfectly matched substrate.”

Researchers Kenneth Johnson, PhD, professor, biochemistry, and member, Institute for Cell and Molecular Biology, and David Taylor, PhD, assistant professor, department of molecular biosciences, The University of Texas at Austin, and colleagues, have identified regions in Cas9 that allow mismatches in DNA binding. This mechanism interferes with on-target activation of the enzyme in CRISPR/Cas9 editing, according to a recent paper published in Nature.

Specifically, they identified a structural loop in the RuvC domain that may reduce off-target DNA cleavage while maintaining rapid on-target cleavage. The research may inform the design of future next-generation CRISPR/Cas9 variants.

CGTLive spoke with Johnson and Taylor to learn more about efforts to improve the speed and fidelity of Cas9 cleavage. They discussed how disrupting the loop structure may be a promising target to achieve this change.

REFERENCE

Bravo, JPK, Liu, MS, Hibshman, GN, et al. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature. Published online March 2, 2022. doi:10.1038/s41586-022-04470-1