Abstract
BackgroundZebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create ‘knockout’ models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies.MethodsTo 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 also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression.ResultsWe compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight 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. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred 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 potentially lasting effect from the microinjection process that requires further investigation.ConclusionOverall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.
Highlights
Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using Clustered regularly inter‐ spaced short palindromic repeats (CRISPR)/Cas9 editing to create ‘knockout’ models
Identification of CRISPR‐induced indels in zebrafish We generated a dataset of experimentally confirmed indels within 14 protein-coding genes from injected NHGRI-1 wild-type zebrafish larvae targeted by 50 guide RNA (gRNA) (2–4 different gRNAs/target gene, assembled through the annealing of crRNA:tracrRNA) (Fig. 1A, Supplementary Tables 1 and 2)
We extracted the percentage of indels using two different tools that deconvolve major mutations and their frequencies within Sanger traces—Tracking of Indels by DEcomposition (TIDE) [34] and Inference from CRISPR Edits (ICE) [35] (Fig. 1B)
Summary
Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas editing to create ‘knockout’ models. New and creative CRISPR-based approaches in zebrafish address biological questions related to developmental processes (e.g., cell-lineage tracing) as well as gene functions (e.g., epigenome editing and targeted mutagenesis, reviewed in [21]) In the latter application, important factors in generating CRISPR gene knockouts include predicting/maximizing ‘on target’ Cas cleavage activity, predicting/minimizing unintended ‘off-target’ editing events, and rapidly detecting small insertions or deletions (indels). Several methods have been devised to experimentally identify off-target cleavage sites (reviewed in [27]), including CIRCLE-Seq [28] and GUIDE-seq [29], that do not depend on prior sequence similarity information These approaches are meant to provide a blind assessment of editing sites but do not necessarily reflect the in vivo activity of on-target activity of the CRISPR/Cas complex
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