Supercoiling twists Cas9 off-target discrimination when nicking and cleaving

Programmed with an RNA guide, Cas9 nuclease directs double-strand DNA cleavage via its two nuclease domains. However, Cas9 sometimes falsely identifies DNA targets by binding and cleaving DNA that does not match its PAM and guide RNA sequence requirements. Cas9s specificity is often affected by DNA topology, as DNA negative supercoiling can increase off-target activity while positive supercoiling can even prevent on-target activity. Such dramatic DNA topological changes routinely occur in cells as a result of transcription and replication, making Cas9s specificity a challenge for gene editing. To determine how Cas9 imparts its specificity across sequences and topologies, we directly mapped kinetic cleavage rates and sites for thousands of relaxed and negatively-supercoiled target sequences via NucleaSeq. We find that: Negative supercoiling can accelerate off-target cleavage a thousand-fold, and shift cleavage sites by two nucleotides. Guide-target mispairs differently impact RuvC and HNH domains, which can lead to topology-dependent nicking by Cas9. Finally, we predict these variations in Cas9 cleavage activity with a biophysical model that accounts for DNA topological state. These efforts expose Cas9s strand-specific off-target cleavage activity and can improve off-target identification for more predictable and safer gene editing. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/715559v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c1d4c5org.highwire.dtl.DTLVardef@77d891org.highwire.dtl.DTLVardef@2b3073org.highwire.dtl.DTLVardef@179d442_HPS_FORMAT_FIGEXP M_FIG C_FIG