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.
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