Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Abstract

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. 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 unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 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. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Review: Plant Genome Editing Targeted by RNA-Guided DNA Endonuclease CRISPR/Cas9

“…moved FRET efficiency to the HNH inactive state [16]. Similar effects were also observed with eSpCas9(1.1) variants.…”

“…The specificity of high-fidelity SpCas9 enzyme was enhanced further by additional mutation D1135E, and two mutations for hydrophobic or stacking interaction of residues L169A and Y450A that interacts with the 5th and 7th PAM proximal nucleotides, respectively [15]. Essentially the same dissociation constants were observed with wild type enzyme, and high fidelity variants eSpCas9(1.1) and SpCas9-hf1 [16].…”

“…A functional approach has been taken to elucidate the interactions of protein domains fluorescently labeled by Cy3/Cy5 dyes and that with target DNA substrate resulting in the enzymatic activation for double-stranded DNA break [16]. moved FRET efficiency to the HNH inactive state [16].…”

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…However, while CRISPR struggles as a standalone therapy, several efforts to minimize off-target cleavage have been reported. Recently, the development of an improved Cas9 variant with enhanced proofreading capacities has extensively reduced off-targeting effects while maintaining the high-cutting efficiency [9]. Additionally, powerful molecules with the ability to inactivate Cas proteins activity, named anti-CRISPR proteins, have been reported to significantly reduce off-targeting edits [89].…”

How Genome Editing could be used in the Treatment of Cardiovascular Diseases

“…While there is concern for off-target effects, in other words, mutations occurring at sites other than the target gene, so far the proof-of-concept studies have found little evidence of this phenomenon in vivo, although the currently used techniques are not sensitive enough to detect mutations occurring in less than one in 10,000 cells. Furthermore, serial innovations with CRISPR-Cas9 are progressively reducing its risk of off-target effects [9][10][11]. Importantly, CRISPR-Cas9 has proven effective in targeting human PCSK9 in authentic human hepatocytes in vivo in a liver-humanized mouse model, suggesting that PCSK9-targeting genome-editing therapies would be similarly effective in live humans [7].…”