ena-STUDY-UNIVERSITY OF CALIFORNIA - DAVIS-29-07-2020-18:23:16:380-2095
CRISPR tools for zebrafish
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. A large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods. To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We compared our experimental in vivo editing 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%). To characterize if commonly used 'mock' CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. From this, while we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundreds of differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the 'mock' injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potential lasting effect from the microinjection process that requires further investigation. Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.
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. A large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods. To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We compared our experimental in vivo editing 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%). To characterize if commonly used 'mock' CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. From this, while we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundreds of differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the 'mock' injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potential lasting effect from the microinjection process that requires further investigation. Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.