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Simple, Efficient CRISPR-Cas9-Mediated Gene Editing in Mice: Strategies and Methods

  • Benjamin E. Low
  • Peter M. Kutny
  • Michael V. WilesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1438)

Abstract

Genetic modification of almost any species is now possible using approaches based on targeted nucleases. These novel tools now bypass previous limited species windows, allowing precision nucleotide modification of the genome at high efficiency, rapidly and economically. Here we focus on the modification of the mouse genome; the mouse, with its short generation time and comparatively low maintenance/production costs is the perfect mammal with which to probe the genome to understand its functions and complexities. Further, using targeted nucleases combined with homologous recombination, it is now possible to precisely tailor the genome, creating models of human diseases and conditions directly and efficiently in zygotes derived from any mouse strain. Combined these approaches make it possible to sequentially and progressively refine mouse models to better reflect human disease, test and develop therapeutics. Here, we briefly review the strategies involved in designing targeted nucleases (sgRNAs) providing solutions and outlining in detail the practical processes involved in precision targeting and modification of the mouse genome and the establishing of new precision genetically modified mouse lines.

Key words

Cas9 CRISPR Genome editing RNA-guided endonucleases Genetic engineering Mouse model Microinjection transgenic Humanization Transgenic sgRNA 

Notes

Acknowledgements

This work was done in close cooperation with The Jackson Laboratory microinjection team. We also fully acknowledge the help and excellent assistance of Cindy Avery and Deb Woodworth for genotyping and animal maintenance, plus discussions regarding CRISPR with Drs. Vishnu Hosur, Wenning Qin, and Gabriele Proetzel. Funding for this work was provided by the National Institutes of Health Grant OD011190 (M.V.W.) and The Jackson Laboratory. We also give our thanks to Addgene for supplying many of the plasmids used in our studies.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Benjamin E. Low
    • 1
  • Peter M. Kutny
    • 1
  • Michael V. Wiles
    • 1
    Email author
  1. 1.The Jackson LaboratoryBar HarborUSA

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