RT Journal Article
SR Electronic
T1 Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 160036
DO 10.1101/160036
A1 Janice S. Chen
A1 Yavuz S. Dagdas
A1 Benjamin P. Kleinstiver
A1 Moira M. Welch
A1 Lucas B. Harrington
A1 Samuel H. Sternberg
A1 J. Keith Joung
A1 Ahmet Yildiz
A1 Jennifer A. Doudna
YR 2017
UL http://biorxiv.org/content/early/2017/08/12/160036.abstract
AB 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 mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust 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.