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J Biol Chem. 2016 Jul 8;291(28):14457-67. doi: 10.1074/jbc.M116.733154. Epub 2016 May 5.

Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.

Author information

1
From the Division of Cellular and Developmental Biology, MCB Department, University of California at Berkeley, Berkeley, California 94705.
2
From the Division of Cellular and Developmental Biology, MCB Department, University of California at Berkeley, Berkeley, California 94705 modzelewski.andrew@berkeley.edu.
3
From the Division of Cellular and Developmental Biology, MCB Department, University of California at Berkeley, Berkeley, California 94705 lhe@berkeley.edu.

Abstract

The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.

KEYWORDS:

CRISPR, Cas9, Genome Editing, Electroporation, RNP; gene knockout; genetics; mouse; ribonuclear protein (RNP); tyrosinase

PMID:
27151215
PMCID:
PMC4938170
DOI:
10.1074/jbc.M116.733154
[Indexed for MEDLINE]
Free PMC Article

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