Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity

Abstract

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

Fig. 1. Multicenter analysis to assess off-target risk in Cas9-edited founder animals using whole genome sequencing (WGS).

Genomic DNA from a subset of C57BL/6N stud males used for embryo production or zygotes that were not treated with Cas9 was used as control DNA. Founders born from Cas9 editing experiments on zygotes from the control stud males or from the same embryo pool comprised the experimental group. Each founder animal was created using a multi-guide strategy to delete a critical exon(s). Founder animals were selected for WGS analysis after confirmation of germline transmission of the expected deletion. The whole-genome sequence analysis pipeline detected single nucleotide variants and small indels as well as potential structural variants. Potential off-target sites were predicted using Cas-OFFinder using permissive parameters and intersected with detected variants to identify putative off-target mutations.

Fig. 2. Summary of variants detected in n = 28 biologically independent control and n = 50 biologically independent Cas9-edited experimental mice.

a Boxplots showing the total number of single nucleotide variants (SNV) and insertion-deletion (indel) variants identified within each experimental group. b Distribution of variants throughout the genome relative to genic sequences. c Zygosity of SNV and indel variants identified. d. Percentage of variants found in sample subsets. For boxplots: Lines within the boxes represent medians, lower edges the 1st quartile, and upper edges the 3rd quartile. The length of the box corresponds to the interquartile range (IQR). Whiskers extend to the minimum or maximum values that fall within 1.5 X IQR of the 1^st and 3^rd quartiles, respectively. Each datapoint represents the number of variants for a given sample. Statistical testing was done using Wilcoxon tests and Bonferroni corrections, evaluated at α = 0.05. HetAlt, heterozygous variant; HomAlt, homozygous variant.

Fig. 3. Detection of potential off-target Cas9 activity in whole genome sequencing data.

a Position of WGS-detected sequence variants associated with predicted off-target sites (N = 26) relative to genes with percentage for each shown in doughnut plot. b Classification of variants associated with WGS-detected predicted off-target sites, associated PAM sequence, and impact of increasing the number of allowed mismatches in Cas-OFFinder predictions from ≤4, 5 or 6. c On target identification of exon deletion at Lpgat1 generated using a four-guide design strategy. Off-target Cas9 activity was associated with guide sequence, g4. Mismatch sites are shown in red lowercase letters. d Primary whole-genome sequence data used to identify off-target site and Sanger sequence validation from founder animal DNA confirming 4-bp deletion in founder. ICE analysis shown below predicts a heterozygous allele frequency (https://www.synthego.com/products/bioinformatics/crispr-analysis). Abbreviations: SV, structural variant; inv, inversion; dup, duplication; del, deletion; SNV, single nucleotide variant; indel, insertion/deletion.

Fig. 4. CIRCLE-seq analysis for select guides.

a Top 10 off-target sites for each guide identified using CIRCLE-seq. The on-target site is marked with an asterisk (*). b Venn diagrams for each guide tested showing overlap between Cas-OFFinder predicted off-targets, CIRCLE-seq and variants detected using whole-genome sequencing (WGS).

Fig. 5. Genetic heterogeneity observed in individual mice of the same isogenic background.

Heatmap shows the percentage of common SNP variants and indels between mice highlighting two major clusters defined by animal production center and mouse substrain used for genetic modification. C57BL/6NCrl (NCrl) mice were used by The Centre for Phenogenomics (TCP) and University of California, Davis (UCD); while C57BL/6NJ (NJ) mice were used by The Jackson Laboratory (JAX) and Baylor College of Medicine (BCM). Sample names are shown on the bottom and right side of figure using the production center abbreviation followed by target gene or substrain background and designation of treatment group either control (C) untreated mice or experimental (E) Cas9-edited founders. Both treatment groups were interspersed with each other consistent with no statistical difference observed between control and experimental mice.