Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment

Nat Commun. 2020 Jul 17;11(1):3596. doi: 10.1038/s41467-020-17418-8.

Abstract

CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / genetics*
  • Gene Editing
  • Humans
  • Mutation
  • RNA, Guide, CRISPR-Cas Systems / genetics

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • DNA