Evaluation of CRISPR gene-editing tools in zebrafish

Abstract

Background 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. 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. 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 also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression. Results We 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. Conclusion Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.

Improved selection of zebrafish CRISPR editing by early next-generation sequencing based genotyping

“…Our findings show that both tools perform well, although a trend to underestimate the actual indel percentage was observed. This finding corresponds with previous literature, where ICE was found to underestimate the presence of edited alleles 22 . Additionally, the performance of the CRISPR-STAT analysis appeared more dependent on the types of indels occurring at the sgRNA cut site, with lower detection rates for small (1–2 BP) indels.…”

“…The selection of sgRNAs with a high cutting efficiency is a crucial step in the generation of a KI model 11 . This can be estimated in silico, although even the most recent computational tools show limited predictive accuracy compared to in vitro assays performed on DNA extracted from injected zebrafish eggs 22 . Most in vitro assays quantify indels in the extracted DNA by separating PCR amplicons with differing lengths 23 , 24 or extracting underlying indel traces from Sanger chromatograms 25 , 26 .…”

Zebrafish as a Model of Cardiac Pathology and Toxicity: Spotlight on Uremic Toxins

“…The CRISPR/Cas9 system is especially useful as a reverse genetic tool because it requires only the enzyme Cas9 and a single guide RNA, which is engineered through the standard DNA cloning technique, to recognize a target site of 20 nucleotides. This approach is efficient for creating mutant-specific loci in zebrafish or for generating mosaic fishes with different mutations [ 97 , 98 ].…”

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

“…However, it is less understood whether resistance is due to mutations that cause changes in the pharmacokinetics and pharmacodynamics of the therapeutic molecules or inherent differences in the pathophysiology of the seizure. The extensive genetic toolkit available in aquatic freshwater vertebrates permits modeling of knockdown [ 101 , 102 , 103 , 104 , 105 , 106 , 107 ], knockout [ 39 , 44 , 46 , 48 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 ], ectopic, and overexpression [ 38 , 117 , 118 , 119 , 120 , 121 ] of seizure-associated genes in combination with metabolic enzymes, transporters, and other proteins required for absorption, distribution, degradation, and excretion of therapeutics. A unique aspect of Xenopus is that their large eggs and embryos can be genetically modified by injecting the embryo on only one side at the two-cell stage, providing an internal control [ 120 ].…”