Mechanisms of improved specificity of engineered Cas9s revealed by single molecule analysis

Abstract

In microbes, CRISPR-Cas systems provide adaptive immunity against invading genetic elements. Cas9 in complex with a guide-RNA targets complementary DNA for cleavage and has been repurposed for wide-ranging biological applications. New Cas9s have been engineered (eCas9 and Cas9-HF1) to improve specificity, but how they help reduce off-target cleavage is not known. Here, we developed single molecule DNA unwinding assay to show that sequence mismatches affect cleavage reactions through rebalancing the internal unwinding/rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily upon mismatch detection. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. DNA interrogation experiments showed that engineered Cas9s require about one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

“…Ultra-stable binding of Cpf1 requires the same extent of sequence match (17 bp PAM-proximal matches) as target cleavage. This contrasts with Cas9, which requires only 9 bp and 16 bp PAM-proximal matches for ultrastable binding and cleavage respectively 18,32,33 . Therefore, Cpf1 can be more sequence specific in experiments involving the use of catalytically dead CRISPR for imaging, tracking and transcription regulation purposes 34 .…”

“…Therefore, Cpf1 can be more sequence specific in experiments involving the use of catalytically dead CRISPR for imaging, tracking and transcription regulation purposes 34 . The binding specificity of engineered Cas9s (eCas9 ) is still much lower than that of Cpf1 33 . Therefore, Cpf1 has the potential to be a better alternative to all current Cas9 variants.…”

“…3), suggesting that shorter RNA-DNA heteroduplexes result in slower conformational changes required for cleavage activation. Previous studies on Cas9 revealed that mismatches alter the kinetics of DNA unwinding, RNA-DNA heteroduplex extension, and nuclease and proof-reading domain movements 19,21,32,33 .…”

Understanding the Molecular Mechanisms of the CRISPR Toolbox Using Single Molecule Approaches

“…Their interaction and the associated changes in molecular conformations can be visualized through FRET between two fluorophores attached to the molecules (Figure A,B). Singh et al , employed smFRET to study the interrogation of donor labeled DNA targets by an acceptor labeled Cas9–RNA. The donor was attached very close to, but just upstream of, PAM so that smFRET restricts the detection window of interrogation events to within ∼10–15 bp of PAM, and as a result, even very transient binding events lasting less than 0.2 s could be confidently detected (Figure C).…”

“…DNA curtain experiments by Sternberg et al and other sm experiments observed that Cas9–RNA remains irreversibly bound to the DNA even after cleavage. Singh et al performed sm experiments to investigate the fate of DNA molecules cleaved by Cpf1 and EngCas9 . While EngCas9s did not release any cleavage products under physiological conditions, Cpf1 released the PAM-distal cleavage product, but not the PAM-proximal product, in the time scale ranging from ∼30 s to 30 min depending on n PD and Cpf1 variants.…”