Rxivist logo

The effect of sequence mismatches on binding affinity and endonuclease activity are decoupled throughout the Cas9 binding site

By Liyang Zhang, H. Tomas Rube, Harmen J. Bussemaker, Miles A. Pufall

Posted 14 Aug 2017
bioRxiv DOI: 10.1101/176255

The CRISPR-Cas9 system is a powerful genomic tool. Although targeted to complementary genomic sequences by a guide RNA (gRNA), Cas9 tolerates gRNA:DNA mismatches and cleaves off-target sites. How mismatches quantitatively affect Cas9 binding and cutting is not understood. Using SelexGLM to construct a comprehensive model for DNA-binding specificity, we observed that 13-bp of complementarity in the PAM-proximal DNA contributes to affinity. We then adapted Spec-seq and developed SEAM-seq to systematically compare the impact of gRNA:DNA mismatches on affinity and endonuclease activity, respectively. Though most often coupled, these simple and accessible experiments identified sometimes opposing effects for mismatches on DNA-binding and cutting. In the PAM-distal region mismatches decreased activity but not affinity, whereas in the PAM-proximal region some reduced-affinity mismatches enhanced activity. This mismatch-activation was particularly evident where the gRNA:DNA duplex bends. We developed integrative models from these measurements that estimate catalytic efficiency and can be used to predict off-target cleavage.

Download data

  • Downloaded 2,964 times
  • Download rankings, all-time:
    • Site-wide: 2,213 out of 100,845
    • In biochemistry: 53 out of 3,444
  • Year to date:
    • Site-wide: 4,284 out of 100,845
  • Since beginning of last month:
    • Site-wide: 7,984 out of 100,845

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

  • 20 Oct 2020: Support for sorting preprints using Twitter activity has been removed, at least temporarily, until a new source of social media activity data becomes available.
  • 18 Dec 2019: We're pleased to announce PanLingua, a new tool that enables you to search for machine-translated bioRxiv preprints using more than 100 different languages.
  • 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!