Rxivist logo

Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 70,616 bioRxiv papers from 308,309 authors.

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy

By Janice S Chen, Yavuz S. Dagdas, Benjamin P. Kleinstiver, Moira M. Welch, Lucas B Harrington, Samuel H. Sternberg, J. Keith Joung, Ahmet Yildiz, Jennifer A. Doudna

Posted 06 Jul 2017
bioRxiv DOI: 10.1101/160036 (published DOI: 10.1038/nature24268)

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 were unknown. 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 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.

Download data

  • Downloaded 4,662 times
  • Download rankings, all-time:
    • Site-wide: 526 out of 70,616
    • In biochemistry: 4 out of 2,118
  • Year to date:
    • Site-wide: 15,758 out of 70,616
  • Since beginning of last month:
    • Site-wide: 18,381 out of 70,616

Altmetric data


Downloads over time

Distribution of downloads per paper, site-wide


PanLingua

Sign up for the Rxivist weekly newsletter! (Click here for more details.)


News