Optimized sgRNA Design to Maximize Activity and Minimize Off-Target Effects of CRISPR-Cas9

Nat Biotechnol. 2016 Feb;34(2):184-191. doi: 10.1038/nbt.3437. Epub 2016 Jan 18.

Abstract

CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Cell Line, Tumor
  • Drug Resistance / genetics
  • Gene Library
  • Genetic Engineering / methods*
  • Genome / genetics
  • Genomics / methods*
  • Humans
  • Mice
  • RNA, Guide / genetics*

Substances

  • RNA, Guide