CRISPR off-target analysis in genetically engineered rats and mice

Abstract

Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.

Figure 1. Off-targets identified from CRISPR projects and in vivo reduction of off-target frequency by re-engineered Cas9.

a, Fraction of projects (animal models) with off-targets. b, Distribution of identified off-targets with respect to known coding genes. c-i, Box-and-whisker plots showing the distribution of 7/21 targets (black dots) and 9/32 off-targets (red dots), sorted by project. Y-axes show fraction of NGS-reads with evidence of Cas9 activity at each locus (one minus fraction of wildtype reads). c, EYFP, n=6 mice. n/d: no data; the EYFP sgRNA targets a mouse knock-in allele and on-target efficiency was analyzed by Sanger sequencing. d, Cflar, n=7 mice. e, Clu, n=5 mice. f, Dhps, n=15 mice. g, Ehmt2, n=14 mice. h, Gsdma, n=10 mice. i, Gsdmc, n=7 mice. All box-and-whisker plots depict min-max range, four quartiles, and center line represents median. Data points depict individual animals. OT: Off-target rank on top 15 list of predicted off-target locations. j, Percent mutation reads at the mouse Pnpla3 target and the originally identified 4 off-targets in vivo. Each datapoint represents one day of microinjection and NGS-analysis of pooled blastocysts. n=3 blastocyst pools comprised of the following number of blastocysts per day and condition; SpCas9_wt (n=45, 32, 56 blastocysts), SpCas9_1.1 (n=50, 51, 65 blastocysts), and SpCas9_HF1 (n=49, 45, 73 blastocysts). Off-target numbering as in Supplementary Fig. 3c. Un-paired two-tailed t-test for on-target means being identical (df=4): wt vs. 1.1, t=0.9025, p=0.42; wt vs. HF1, t=0.4260, p=0.69; 1.1 vs. HF1, t=0.3259, p=0.76. k, Percent mutation reads at the rat Map3k14 target and originally identified off-target in vivo. Each datapoint represents NGS-analysis of one rat embryo, SpCas9_wt n=17, SpCas9_1.1 n=30, SpCas9_HF1 n=24 embryos. Off-target numbering as in Supplementary Fig. 3g. Un-paired two-tailed t-test for on-target means being identical: wt vs. 1.1, df=45, t=3.488, p=0.0011; wt vs. HF1, df=39, t=10.66, p<0.0001; 1.1 vs. HF1, df=52, t=6.994, p<0.0001. For a, b, center line represents mean and error bars show SEM.

Figure 2. TEG-seq is a good predictor of in vivo activity.

a, Radar plots showing the distribution of on- and off-target activity across the target and 43 off-target loci identified by whole-genome sequencing. Off-target numbers are indicated on the periphery. Data from two embryos is shown (185, least number of off-targets, 231, most off-targets). Percent mutation read quartiles are indicated by grey circles. b, Correlation between ampli-seq mutation frequency in Neuro-2a cells and average embryo mutation frequency (n=119 loci, 118 off-targets, black dots, and Pnpla3 target, red dot). r² value (coefficient of determination) is indicated (correlation coefficient r=0.9225. Two-tailed t-test: r value significantly different from zero, p<0.0001). c, box-and-whisker plot depicting number of off-targets with at least 10% mutation efficiency per embryo, n=10 mouse embryos. Plot shows min-max range, four quartiles, and center line represents median.