DNA-binding-domain fusions enhance the targeting range and precision of Cas9

Nat Methods. 2015 Dec;12(12):1150-6. doi: 10.1038/nmeth.3624. Epub 2015 Oct 19.


The CRISPR-Cas9 system is commonly used in biomedical research; however, the precision of Cas9 is suboptimal for applications that involve editing a large population of cells (for example, gene therapy). Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but they often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site in the human genome over a large cohort of treated cells. Here we show that by fusing a programmable DNA-binding domain (pDBD) to Cas9 and attenuating Cas9's inherent DNA-binding affinity, we were able to produce a Cas9-pDBD chimera with dramatically improved precision and an increased targeting range. Because the specificity and affinity of this framework can be easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Cas Systems / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA Cleavage
  • DNA-Binding Proteins / genetics*
  • Gene Targeting* / methods
  • HEK293 Cells
  • Humans
  • RNA, Guide / genetics
  • Transcriptional Activation
  • Transfection
  • Zinc Fingers / genetics


  • CRISPR-Associated Proteins
  • DNA-Binding Proteins
  • RNA, Guide

Associated data

  • SRA/SRP063361