Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq (original) (raw)

  1. Sojung Kim1,2,
  2. Sunghyun Kim1,
  3. Jeongbin Park3 and
  4. Jin-Soo Kim1,2
  5. 1Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea;
  6. 2Department of Chemistry, Seoul National University, Seoul 151-747, South Korea;
  7. 3Department of Chemistry, Hanyang University, Seoul 133-791, South Korea
  8. Corresponding author: jskim01{at}snu.ac.kr

Abstract

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.

Footnotes

This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.