Targeting G-quadruplex Forming Sequences with Cas9

Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting editing specific DNA sequences. If the target sequences are prone to folding into noncanonical secondary structures, such as G-quadruplex (GQ), conformational states activity of CRISPR–Cas9 complex may be influenced, but impact has not been assessed. Using single molecule FRET, we investigated structural characteristics formed by DNA, which contains a potentially GQ forming sequence (PQS) in either or nontarget strand (TS NTS). We observed different dynamics depending stability position PQS. When PQS was NTS, evidence for formation both weak stable GQs. This is consistent with R-loop between TS crRNA releasing NTS from Watson–Crick pairing facilitating structure it. TS, adequate maintain unfolded state moderate high stability. The heterogeneity within dsDNA strongly depended whether NTS. propose these variations structures functional implications Cas9 activity.