Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells

Abstract

Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

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.…”

“…Potential offtarget sequences were identified by digestion of iPSC-derived, cell-free genomic DNA with CRISPR-Cas9 followed by WGS. Sequencing reads of CRISPR-Cas9-digested genomic DNA are vertically aligned at on-/off-target sites in IGV viewer 25,27 . By contrast, undigested genomic sites are aligned in a staggered manner at those loci.…”

“…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 vivo gene therapy potentials of CRISPR-Cas9

“…Therefore, genome-wide unbiased methods such as GUIDE-seq and Digenome-seq should be harnessed to provide a comprehensive profile of the off-target events. [20][21][22] As off-target genome editing may incur unwanted consequences including malignancies, they should be precisely profiled and reduced to almost nil if applied to human gene therapy. Significant advances have been made recently to reduce the off-target effects, such as double nicking by Cas9 nickases, 23 optimizing sgRNA design 24,25 and reconstruction of Cas9 nuclease.…”