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bioRxiv · 10.1101/2023.08.05.552134

Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications

Hu, C.·Myers, M. T.·Zhou, X.·Hou, Z.·Lozen, M. L.·Zhang, Y.·Ke, A.
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Abstract

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets, and the nuclease-helicase Cas3 to degrade them. Among seven subtypes, Type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here we use four cryo-electron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling efficient NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9, that strongly inhibit N. lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through direct competition, whereas AcrIC9 achieves so through allosteric inhibition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for controllable Type I CRISPR genome engineering.

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Hu, C., Myers, M. T., Zhou, X., Hou, Z., Lozen, M. L., Zhang, Y., Ke, A.. 2023-08-06. Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications. https://doi.org/10.1101/2023.08.05.552134

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