Abstract
Several strategies have been developed to generate targeted gene disruptions in zebrafish.
Here we developed a simple targeted gene inactivation strategy in zebrafish using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. By injecting two simple in vitro-synthesized components [Cas9 mRNA and single guide (sgRNA)] into one-cell-stage embryos, mutations of the target gene could be efficiently generated. We used a codon-optimized version of Cas9 to improve its translation efficiency in zebrafish. In addition, we designed a cloning-free strategy to facilitate the synthesis of sgRNA. The system allows biallelic inactivation of multiple genes simultaneously by co-injecting a mix of sgRNAs with a single Cas9 construct. This flexible strategy of gene inactivation provides an efficient way to interrogate gene functions and genetic interactions in zebrafish.
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Acknowledgements
We thank Shawn Burgess for communicating unpublished data. This work was supported by R01DK088686 (to W.C.) from the NIH.
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Yin, L., Jao, LE., Chen, W. (2015). Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System. In: Fiedler, L. (eds) VEGF Signaling. Methods in Molecular Biology, vol 1332. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2917-7_16
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DOI: https://doi.org/10.1007/978-1-4939-2917-7_16
Publisher Name: Humana Press, New York, NY
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