Efficient mouse genome engineering by CRISPR-EZ technology

Andrew J. Modzelewski, Sean Chen, Brandon J. Willis, Kevin C K Lloyd, Joshua Wood, Lin He

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

Original languageEnglish (US)
Pages (from-to)1253-1274
Number of pages22
JournalNature Protocols
Volume13
Issue number6
DOIs
StatePublished - Jun 1 2018

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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