Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

Genome Res. 2016 Mar;26(3):406-15. doi: 10.1101/gr.199588.115. Epub 2016 Jan 19.


We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • CRISPR-Cas Systems*
  • Gene Targeting*
  • Genome, Human*
  • Genomics* / methods
  • High-Throughput Nucleotide Sequencing
  • Humans
  • INDEL Mutation
  • Mutation Rate
  • Nucleotide Motifs
  • Position-Specific Scoring Matrices
  • Protein Binding
  • Reproducibility of Results