A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

Abstract

A conformational checkpoint of Cas9 enables on-target cleavage while blocking access to the active state at off-target sites.

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…DNA binding assays were conducted in 1X binding buffer without MgCl 2 + 1 mM EDTA at room temperature for 2 hr. DNA-bound complexes were resolved on 8% native PAGE (0.5X TBE + 1 mM EDTA, without MgCl 2 ) at 4 °C, as previously described 10 . Experiments were replicated at least three times, and presented gels are representative results.…”

“…Sample preparation was performed as previously described 10 . Briefly, the glass surface was pre-blocked with casein (10 mg/ml) for 10 min.…”

“…Whereas SpCas9 HNH stably populated the active state with on-target and mismatched substrates as observed by steady-state smFRET (Figure 1d), only ~32% of SpCas9-HF1 HNH molecules occupied the HNH active state (E FRET = 0.97) with an on-target substrate, with the remaining ~68% trapped in the inactive intermediate state (E FRET = 0.45) (Figure 1e). Of the dynamic molecules (~36% of all smFRET traces) observed for SpCas9-HF1 HNH , kinetics analysis further revealed that the HNH transition rate from the inactive to active states was ~8-fold slower compared to that of WT SpCas9 HNH (~3% dynamic molecules 10 ) (Extended Data Figure 3). However, when SpCas9-HF1 HNH was bound to a substrate with a single mismatch at the PAM-distal end (20-20 bp mm), stable docking of the HNH nuclease was entirely ablated (Figure 1e).…”

“…High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown 5–9 . Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state 10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence.…”

“…It was previously shown that the HNH domain stably docks in its active state with an on-target substrate, but becomes loosely trapped in a catalytically-inactive conformational checkpoint when bound to mismatched targets 10,12 . We therefore hypothesized that SpCas9-HF1 and eSpCas9(1.1) variants may employ a more sensitive threshold for HNH domain activation to promote off-target discrimination.…”