Sensitive detection of off-target effects is important for translating CRISPR–Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR–Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR–Cas9.
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This work was supported by a National Institutes of Health (NIH) Director's Pioneer Award (DP1 GM105378), NIH R35 GM118158, and NIH R01 GM107427 (to J.K.J.); and the Jim and Ann Orr Research Scholar Award (to J.K.J.).
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Integrated supplementary information
Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem loop adapter 1. Covalently closed DNA molecules with stem-loop adapters ligated to both ends are selected for by treatment with a mixture of Lambda exonuclease and E. coli Exonuclease I, and then treated with Cas9-sgRNA complex. Cleaved molecules will have a newly available end for subsequent ligation of stem-loop adapter 2. Ligation of both stem loop adapters provides required 5’ and 3’ sequences for PCR and high-throughput sequencing.
Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-loop adapters. Covalently closed DNA molecules with stem-loop adapters ligated to both ends are selected for by treatment with a mixture of Lambda exonuclease and E. coli Exonuclease I. 4 bp overhangs are released with a mixture of USER enzyme and T4 PNK, and DNA molecules are circularized at low concentrations favoring intramolecular ligation. Unwanted linear DNA is degraded with Plasmid-Safe ATP-dependent DNase. Circular DNA is treated with Cas9–sgRNA complex and cleaved, linearized DNA is ligated to sequencing adapters and amplified for high-throughput sequencing.
Supplementary Figure 3 Optimization of in vitro circularization conditions with uracil-containing stem loop adapters and a PCR amplicon.
Qiaxcel capillary electrophoretic traces of intramolecular ligation, exonuclease treatment, and restriction enzyme digestion. The observed electrophoretic mobility shift is consistent with circularization. An exonuclease-resistant population of circular DNA molecules is observed after Plasmid-Safe treatment. Digestion with BamHI restriction enzyme linearizes the circularized DNA and results in the expected shift in mobility.
Supplementary Figure 4 Comparison of CIRCLE-seq and covalently closed linear stem-loop strategies for identifying nuclease-induced off-target effects.
(a) Scatterplot of read counts for linear stem-loop and circular (CIRCLE-seq) methods for detecting Cas9 nuclease-induced off-target sites for a sgRNA targeted against VEGFA site 1. (b) Venn diagram showing overlap of sites detected by CIRCLE-seq and alternative linear stem-loop method.
Scatterplots of CIRCLE-seq read counts between two independent CIRCLE-seq libraries prepared from the same source of genomic DNA (human U2OS cells) for sgRNAs targeted against EMX1 and VEGFA site 1.
(a) Venn diagram showing intersections of off-target sites of Cas9 and a sgRNA targeted against the HBB gene detected by CIRCLE-seq (blue) and Digenome-seq (clear). (b) CIRCLE-seq reads observed at 3 sites that are called by Digenome-seq but not CIRCLE-seq. Integrated Genome Viewer (IGV) plots showing supporting CIRCLE-seq read alignments mapped to human reference genome (GrCh37). Reads mapping to the reverse strand are colored in blue, reads mapping to the forward strand are colored in red. (c) Barplot of Digenome-seq start mapping read counts at off-target cleavage positions identified by CIRCLE-seq but not called by Digenome-seq for nuclease-treated (red) and control (blue) HAP1 genomic DNA. (d) Plots comparing mapping of sequencing reads for CIRCLE-seq and Digenome-seq at the on-target site of a sgRNA targeted to the HBB locus. Both nuclease-treated and control samples are shown. A thin grey line indicates expected cleavage site position; read coverage for forward reads is colored in red, and reverse reads in blue. (e) CIRCLE-seq start mapping position plot at the on-target site for the HBB sgRNA used in (c). (+) strand mapping reads are colored in blue, (-) strand mapping reads are colored in green.
Number of mismatches in CIRCLE-seq detected off-target sites relative to the intended target site of sgRNAs targeted against standard sites in HEK293 & U2OS cells, repetitive sites in HEK293 & U2OS cells, and sites in K562 cells.
Supplementary Figure 8 Venn diagrams showing intersection of CIRCLE-seq and GUIDE-seq detected genomic off-target cleavage sites.
CIRCLE-seq sites are indicated in blue and GUIDE-seq sites with clear circles. The top six comparisons are for sgRNAs targeted against standard genomic sites, and the bottom four comparison are targeted against more repetitive sites.
Supplementary Figure 9 Venn diagrams showing overlap between sets of off-target cleavage sites detected between CIRCLE-seq, GUIDE-seq, and HTGTS.
CIRCLE-seq (solid blue) detects virtually all off-target cleavage sites detected by both GUIDE-seq (hatched blue) and HTGTS (clear).
Normalized GUIDE-seq read counts plotted against normalized CIRCLE-seq reads grouped by mismatch numbers between 0 and 6.
Percentage of unique cleavage sites that can be found using a reference-independent site discovery algorithm, for CIRCLE-seq experiments performed with sgRNAs targeting non-repetitive sites in HEK293 (red), K562 (green), and U2OS genomic DNA (blue).
Supplementary Figure 12 Effects of titrating Cas9 protein concentration on in vitro cleavage efficiency and number of CIRCLE-seq sites detected.
(a) Barplot of percent in vitro cleavage of a targetsite containing PCR amplicon by Cas9 at different concentrations. (b) Number of sites detected by CIRCLE-seq at different concentrations of Cas9:sgRNA complex. Validated sites include both those detected by GUIDE-seq and by confirmatory targeted tag sequencing.
Supplementary Figures 1–12, Supplementary Notes 1–2 and Supplementary Table 1. (PDF 5708 kb)
List of all CIRCLE-seq detected off-target sites. (XLSX 640 kb)
List of CIRCLE-seq read counts and HTGTS scores for off-target sites detected for Cas9 and gRNAs targeted against EMX1 and VEGFA site 1. (XLSX 66 kb)
Deep sequencing read counts for targeted tag integration sequencing of off-target cleavage sites of Cas9 and gRNAs targeted against EMX1 and VEGFA site 1. (XLSX 53 kb)
Listing of cell-type specific SNPs in protospacer or PAM of off-target cleavage sites detected by CIRCLE-seq. (XLSX 52 kb)
Primers used in target tag integration sequencing. (XLSX 18 kb)
Supplementary Protocol: CIRCLE-seq Library Preparation. (PDF 581 kb)
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Tsai, S., Nguyen, N., Malagon-Lopez, J. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat Methods 14, 607–614 (2017). https://doi.org/10.1038/nmeth.4278
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