Structural basis of supercoiling-induced CRISPR-Cas9 off-target activity

CRISPR–Cas9 is a powerful genome-editing tool1, but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling ((−)SC) has been implicated in off-target activity, but a molecular-level understanding is lacking. Here, using (−)SC DNA minicircles, we observe supercoiling-driven structural defects in the DNA that are resolved by Cas9 binding. Cryo-electron microscopy structures of Cas9 bound in both the on-target and off-target configurations highlight that the Cas9 HNH domain is poised in a more catalytically competent conformation. New DNA–RNA mismatch geometries are accommodated across the protospacer and structural plasticity in the protospacer adjacent motif distal region of the protospacer is topology dependent. Together, our study reveals the molecular basis for (−)SC-induced Cas9 targeting and provides a framework for the design of next-generation high-fidelity CRISPR effectors with topological context. Cas9 structures explain topology sensing and off-target activation. DNA minicircles are versatile substrates for DNA topology studies29,30,31,32. Using splinted ligation (Extended Data Fig. 1a), we have generated 126 bp DNA minicircles containing the well-characterized EMX1-1 target sequence (Fig. 1a,b) and two off-t... [42126 chars]