Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 60,239 bioRxiv papers from 267,831 authors.
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
Janice S Chen,
Yavuz S. Dagdas,
Benjamin P. Kleinstiver,
Moira M. Welch,
Lucas B Harrington,
Samuel H. Sternberg,
J. Keith Joung,
Jennifer A Doudna
Posted 06 Jul 2017
bioRxiv DOI: 10.1101/160036 (published DOI: 10.1038/nature24268)
Posted 06 Jul 2017
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.
- Downloaded 4,602 times
- Download rankings, all-time:
- Site-wide: 453 out of 60,239
- In biochemistry: 4 out of 1,746
- Year to date:
- Site-wide: 18,169 out of 60,239
- Since beginning of last month:
- Site-wide: 36,450 out of 60,239
Downloads over time
Distribution of downloads per paper, site-wide
- Top preprints of 2018
- Paper search
- Author leaderboards
- Overall metrics
- The API
- Email newsletter
- 21 May 2019: PLOS Biology has published a community page about Rxivist.org and its design.
- 10 May 2019: The paper analyzing the Rxivist dataset has been published at eLife.
- 1 Mar 2019: We now have summary statistics about bioRxiv downloads and submissions.
- 8 Feb 2019: Data from Altmetric is now available on the Rxivist details page for every preprint. Look for the "donut" under the download metrics.
- 30 Jan 2019: preLights has featured the Rxivist preprint and written about our findings.
- 22 Jan 2019: Nature just published an article about Rxivist and our data.
- 13 Jan 2019: The Rxivist preprint is live!