Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create knockout models. Due to the large number of available tools to design CRISPR assays and diversity of theories/model systems they were originally built on, we sought to systematically compare computational and empirical approaches for predicting gene-editing efficacy in zebrafish. We subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes and assayed individual editing efficiencies. We compared our experimental in vivo efficiencies in mosaic G0 embryos with those predicted by seven commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (<1%). Moreover, understanding that recent segmental duplications in the zebrafish genome could exacerbate CRISPR targeting of individual genes, we cataloged these loci and have made them available as a resource. Lastly, we assessed the transcriptome of negative mock control CRISPR larvae injected with Cas9 enzyme or mRNA with no gRNA using RNA-seq and identified differentially expressed genes with high variability between injections. Using these same data, we discovered on average ~60 putative somatic mosaic frameshift mutations impacting genes per pool of injected larvae, potentially due to background cutting of DNA with Cas9 in the absence of gRNA. To verify this previously unreported phenomenon in zebrafish, we validated seven of twelve genes tested carrying low frequency mosaic somatic mutations in the genomes of a separate batch of injected larvae. These results suggest that negative control embryos may carry mutations within genes leading to spurious phenotypes. Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments. AUTHOR SUMMARYZebrafish have proven to be a powerful model organism for the functional characterization of genes. Development of new workflows targeting individual or multiple genes simultaneously require a thorough understanding of the advantages and limitations of current available methods for CRISPR-editing in zebrafish. Here, we systematically evaluated on- and off-target efficiencies prediction methods of 50 gRNAs by experimentally testing their CRISPR cutting efficiencies in embryos. Moreover, we performed a global assessment of duplicated portions of the zebrafish genome, providing a powerful resource for the design of future CRISPR assays. Lastly, we evaluated the possibility that spurious editing occurs in samples injected with the Cas9 nuclease without a gRNA, which are commonly used as a baseline control. This analysis revealed high variability in gene expression and the presence of frameshift variants in larvae injected solely with Cas9, suggesting that additional caution should be taken when using these samples as baseline controls in functional characterizations of genes.