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Rapid reverse genetic screening using CRISPR in zebrafish.

ABSTRACT: Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. We used engineered CRISPR (clustered regularly interspaced short palindromic repeats) to efficiently mutate specific loci in zebrafish (Danio rerio) and screen for genes involved in vertebrate biological processes. We found that increasing CRISPR efficiency by injecting optimized amounts of Cas9-encoding mRNA and multiplexing single guide RNAs (sgRNAs) allowed for phenocopy of known mutants across many phenotypes in embryos. We performed a proof-of-concept screen in which we used intersecting, multiplexed pool injections to examine 48 loci and identified two new genes involved in electrical-synapse formation. By deep sequencing target loci, we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system.

SUBMITTER: Shah AN

PROVIDER: S-EPMC4667794 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Rapid reverse genetic screening using CRISPR in zebrafish.

Shah Arish N AN Davey Crystal F CF Whitebirch Alex C AC Miller Adam C AC Moens Cecilia B CB

Nature methods 20150413 6

Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. We used engineered CRISPR (clustered regularly interspaced short palindromic repeats) to efficiently mutate specific loci in zebrafish (Danio rerio) and screen for genes involved in vertebrate biological processes. We found that increasing CRISPR efficiency by injecting optimized amounts of Cas9-encoding mRNA and multiplexing single guide RNAs (sgRNAs) allowed for phenocopy of ...[more]

PMID: 25867848

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Project description:BackgroundWith the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification) provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications.FindingsWe employed a dual-colour RT-MLPA probe design for chemical synthesis of probe pairs for 34 genes involved in Toll-like receptor signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. In addition, six probe pairs were included for reference genes unaffected by infections in zebrafish. First, we established assay conditions for adult zebrafish infected with different strains of Mycobacterium marinum causing acute and chronic disease. Addition of competitor oligonucleotides was required to achieve peak heights in a similar range for genes with different expression levels. For subsequent analysis of embryonic samples it was necessary to adjust the amounts of competitor oligonucleotides, as the expression levels of several genes differed to a large extent between adult and embryonic tissues. Assay conditions established for one-day-old Salmonella typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum-infected larvae. RT-MLPA results were compared with results of previous transcriptome analyses and with real-time PCR data, demonstrating a good correlation between all expression analysis methods.ConclusionsThe RT-MLPA assay developed in this study provides a rapid, cheap, and robust analysis tool for simultaneous quantification of a set of 34 innate immune response genes. With adjustment of conditions, the assay is suitable for infection studies in both adult and embryonic zebrafish. Application of RT-MLPA will facilitate high-throughput screening of immune responses in the zebrafish model.

Dataset Information

Rapid reverse genetic screening using CRISPR in zebrafish.

Publications

Rapid reverse genetic screening using CRISPR in zebrafish.

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