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Open Access Publications from the University of California

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

  • Author(s): Chen, Janice S
  • Dagdas, Yavuz S
  • Kleinstiver, Benjamin P
  • Welch, Moira M
  • Sousa, Alexander A
  • Harrington, Lucas B
  • Sternberg, Samuel H
  • Joung, J Keith
  • Yildiz, Ahmet
  • Doudna, Jennifer A
  • et al.

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The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. 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 are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state 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 design 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.

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