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

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 falsepositive, 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.

Correction of a pathogenic gene mutation in human embryos

“…Therefore, we conducted a comprehensive, whole-genome In the S-phase-injected group, each mosaic embryo (green) contained blastomeres with different genotypes. For source data, see Supplementary Table 4. sequencing (WGS) analysis of the patient's genomic DNA by digested genome sequencing (Digenome-seq) 25,26 . Potential offtarget sequences were identified by digestion of iPSC-derived, cell-free genomic DNA with CRISPR-Cas9 followed by WGS.…”

“…By contrast, undigested genomic sites are aligned in a staggered manner at those loci. In addition, improved Digenome-seq provides DNA cleavage scores for potential off-target sites based on alignment patterns of sequence reads 26 . Digested iPSC DNA produced uniform cleavage patterns at both on-target and potential off-target sites (Extended Data Fig.…”

“…Genomic DNA was isolated from patient iPSCs using a DNeasy Tissue Kit (Qiagen). Digenome-seq was performed as described 25,26 . In brief, 20 μ g genomic DNA was cleaved by incubating recombinant Cas9 protein (16.7 μ g) and in vitro transcribed sgRNA (12.5 μ g) in 1× NEB buffer 3.1(100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μ g/ml BSA, pH 7.9) at 37 °C for 3 h. Cas9-and sgRNA-treated genomic DNA was treated with 50 μ g/ml RNase A (Sigma Aldrich) at 37 °C for 30 min, and purified with a DNeasy Tissue Kit (Qiagen).…”

“…In brief, 20 μ g genomic DNA was cleaved by incubating recombinant Cas9 protein (16.7 μ g) and in vitro transcribed sgRNA (12.5 μ g) in 1× NEB buffer 3.1(100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μ g/ml BSA, pH 7.9) at 37 °C for 3 h. Cas9-and sgRNA-treated genomic DNA was treated with 50 μ g/ml RNase A (Sigma Aldrich) at 37 °C for 30 min, and purified with a DNeasy Tissue Kit (Qiagen). WGS and Digenome sequencing were performed as described previously 25,26 . In brief, 1 μ g genomic DNA was fragmented and ligated with adaptors using TruSeq DNA libraries.…”

“…In vitro DNA cleavage sites were identified computationally using a DNA cleavage scoring system described previously 26 . Indel frequencies of 23 genomic loci with DNA cleavage score above the 0.1 cutoff value were individually examined in individual blastomeres by targeted deep sequencing.…”