CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets

Abstract

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.

Figure 1. Overview of CIRCLE-seq methods for detection of genome-wide CRISPR-Cas9 nuclease cleavage

Schematic overview of the circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) method. Genomic DNA is sheared and circularized by ligation of stem-loop adapters, nicking of stem-loop regions to expose 4 nt palindromic overhangs, and intramolecular ligation. Undesired linear DNA molecules are degraded away by exonuclease treatment. Circular DNA molecules containing a Cas9 cleavage site (red) can be subsequently linearized with Cas9, releasing newly cleaved DNA ends for adapter ligation, PCR amplification, and paired-end high-throughput sequencing. Each pair of reads generated by Cas9 cleavage contains complete sequence information for a single off-target site.

Figure 2. Comparisons of CIRCLE-seq with cell-based GUIDE-seq and HTGTS methods

(a) Histogram showing the number of sites identified exclusively by CIRCLE-seq (blue) and by both CIRCLE-seq and GUIDE-seq (magenta) for gRNAs designs toward standard and more challenging repetitive targets (b) Manhattan plots of CIRCLE-seq detected off-target sites, with bar heights representing CIRCLE-seq read count (normalized to site with highest read count) and organized by chromosomal position. (c) Histogram showing the number of sites detected exclusively by CIRCLE-seq (blue) or by both CIRCLE-seq and HTGTS (yellow).

Figure 3. CIRCLE-seq detected off-target cleavage sites can also be cleaved in human cells

(a) Stem-leaf plot of CIRCLE-seq read counts for 10 gRNAs previously analyzed by GUIDE-seq. The on-target site is shown as a green dot, and off-target sites detected by GUIDE-seq are shown as red dots. (b) Schematic overview of the targeted tag sequencing approach. Primers are designed to amplify genomic regions flanking nuclease-induced DSBs from genomic DNA of cells treated with nuclease and double-stranded oligodeoxynucleotide (dsODN) tag. (c-d) Targeted tag integration frequencies at control off-target sites detected by both CIRCLE-seq and GUIDE-seq (upper part of panel) and off-target sites detected by CIRCLE-seq but not GUIDE-seq) for gRNAs targeted to EMX1 and VEGFA site 1. Off-target sites are ordered top to bottom by CIRCLE-seq read count with mismatches to the intended target sequence indicated by colored nucleotides. Observed tag integration frequencies observed for control (blue) and nuclease-treated (red) cells are plotted on a log scale. (e) Pie charts showing fractions of CIRCLE-seq sites analyzed that are also detected by targeted tag sequencing. (f) Plots of integration positions observed by targeted tag sequencing. PAM bases are the last three nucleotides from the right. Integrations occur at positions proximal to the location of the predicted DSB (three base pairs away the PAM).

Figure 4. Using CIRCLE-seq to assess the impacts of personalized SNPs on off-target site analysis

(a) Scatterplots of CIRCLE-seq read counts from experiments performed on genomic DNA from two different cell types. Sites with non-reference genetic variation in only one cell type are highlighted in red, while those with non-reference variation in both cell types are highlighted in blue. (b) Examples of allele-specific CIRCLE-seq read counts at off-target sites with non-reference genetic variation. Mismatches to the intended target sequence are indicated with colored nucleotides, while matching bases are indicated with a dot. The base position harboring the differential genetic change between cell types is indicated with a small arrow. (c) Proportion of CIRCLE-seq off-target sites where non-reference genetic variation was identified in genotyped individuals from the 1000 Genomes Project: African (AFR), Ad Mixed American (AMR), East Asian (EAS), European (EUR), and Southeast Asian (SAS) superpopulations, and a combined population average. (d) Histogram showing distribution of CIRCLE-seq off-target sites by numbers of mismatches in reference human genome sequence (red) and in 1000 Genomes Project data-derived off-target site haplotypes (blue). (e) Proportion of 1000 Genomes Project-derived haplotypes with increased (blue), decreased (red), or same (grey) numbers of mismatches in off-target sites identified by CIRCLE-seq.