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Rapid reverse genetic screening using CRISPR in zebrafish

Nature Methods volume 12, pages 535540 (2015) | Download Citation

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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.

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Acknowledgements

We thank R. Garcia for superb animal care, the Moens lab for discussion and editing, J. Spiewak and the Parichy lab at the University of Washington for slc24a5 mutant embryos, S. Peterson and the Postlethwait lab at the University of Oregon for advice on sgRNA cloning, and I. Phelps and D. Doherty for help and access to the MiSeq machine. Funding was provided by the National Institutes of Health (R01HD076585 and R21NS076950 to C.B.M. and K99NS085035 to A.C.M.).

Author information

Author notes

    • Adam C Miller
    •  & Cecilia B Moens

    These authors jointly directed this work.

Affiliations

  1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

    • Arish N Shah
    • , Crystal F Davey
    • , Alex C Whitebirch
    • , Adam C Miller
    •  & Cecilia B Moens

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Contributions

A.N.S., C.F.D., A.C.W. and A.C.M. performed experiments, acquired and quantified data, and generated images for publication. A.C.M., A.N.S. and C.B.M. wrote the manuscript. All authors edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Adam C Miller or Cecilia B Moens.

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DOI

https://doi.org/10.1038/nmeth.3360

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