Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications

Nucleic Acids Res. 2018 Feb 16;46(3):1375-1385. doi: 10.1093/nar/gkx1268.

Abstract

Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Base Composition
  • Base Sequence
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Gene Editing / methods*
  • Gene Targeting / methods*
  • HEK293 Cells
  • Humans
  • Leukocytes / cytology
  • Leukocytes / metabolism
  • Nucleic Acid Conformation
  • RNA, Guide, CRISPR-Cas Systems / chemistry
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes