title: Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease.
abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted nonhomologous end joining and homology-directed repair mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.
authors.forename: Wenning
authors.initials: W
authors.lastname: Qin
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Dion
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kutny
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.
authors.forename: Yingfan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.
authors.forename: Albert W
authors.initials: AW
authors.lastname: Cheng
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.
authors.forename: Nathaniel L
authors.initials: NL
authors.lastname: Jillette
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.
authors.forename: Ankit
authors.initials: A
authors.lastname: Malhotra
authors.affiliation: Human Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Aron M
authors.initials: AM
authors.lastname: Geurts
authors.affiliation: Human Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Yi-Guang
authors.initials: YG
authors.lastname: Chen
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100090, China wanghaoyi@ioz.ac.cn.
authors.forename: Haoyi
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D018274:Electroporation; D004720:Endonucleases / Q000235:genetics*; D005260:Female; D018390:Gene Targeting; D056426:Genetic Loci; D016678:Genome; D023281:Genomics; D054643:INDEL Mutation; D051379:Mice; D008969:Molecular Sequence Data; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D015053:Zygote / Q000378:metabolism*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D004720:Endonucleases
keywords: CRISPR; Cas9; electroporation; mouse zygote
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