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

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…