Whole exome sequencing in two generations of CRISPR-Cas9 edited zebrafish. (A) The experimental design generates a single clutch of ∼200 embryos from a founder pair of parents from the ZDR laboratory strain of wild-type zebrafish. The embryos were randomly assigned to four experimental arms: uninjected controls, Cas9 injected controls, sgRNA injected controls, and Cas9 + sgRNA gene edited samples. A total of 52 embryos were sampled for DNA extraction and sequencing at 4 dpf in the F0 generation (2 uninjected, 2 Cas9 injected, 2 sgRNA injected across 6 different sgRNAs targeting 3 genes for a total of 12 embryos, and 6 CRISPR-Cas9 embryos per sgRNA guide for a total of 36 edited individuals). Additional embryos for each condition were injected concurrently, but raised to adulthood. The F0 in-cross from pairs edited with the smchd1 high efficiency guide generated F1 progeny for further sequencing: We sampled offspring from 4 uninjected, 4 Cas9 injected, 4 sgRNA injected, and 4 CRISPR-Cas9 injected embryos for a total of 16 F1 exomes. (B) The first round of exome sequencing (F0 and parents) generated a consistent read depth averaging 76x coverage. (C) The second round of exome sequencing (F1) generated a consistently higher read depth averaging 115x coverage. The smchd1 edited individuals are also sequenced to a higher depth than the uninjected controls (p < 0.05). (D) After sequencing quality control and alignment, variant calling was performed with both somatic and germline callers to identify candidate de novo mutations.

Counts of candidate de novo mutations in control and edited individual zebrafish embryos. Variants persisting after filtering and with an allele frequency ≥0.3 are not significantly different between control and CRISPR-Cas9 edited groups (N = 68). (A) Predicted counts by VarScan2. (B) Unambiguous heterozygous variants determined by visual inspection of VarScan2 calls in IGV (C) Subset of predicted variants detected by both variant callers.

Acknowledgments
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