Dataset Information

Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.

ABSTRACT: The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Unexpected or unknown transcripts, generated through alternative splicing, may impede the generation of successful loss-of-function mutants. To create null or null-like loss-of-function mutant zebrafish, we employed simultaneous multiple gRNA injection into single-cell stage embryos. We demonstrated that DNA composed of multiple exons, up to 78kb in length, can be deleted in the smarca2 gene locus. Additionally, two different genes (rnf185 and rnf215) were successfully mutated in F1 fish with multiple exon deletions using this multiplex gRNA injection strategy. We expect this approach will be useful for knock-out studies in zebrafish and other vertebrate organisms, especially when the phenotype of a single gRNA-induced mutant is not clear.

SUBMITTER: Kim BH

PROVIDER: S-EPMC7056962 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.

Kim Brian H BH Zhang GuangJun G

G3 (Bethesda, Md.) 20200305 3

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Une ...[more]

PMID: 31915253

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Project description:Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.

Dataset Information

Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.

Publications

Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.

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