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Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1


Dumeau, Charles-Etienne; Monfort, Asun; Kissling, Lucas; Swarts, Daan C; Jinek, Martin; Wutz, Anton (2019). Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1. Transgenic research, 28(5-6):525-535.

Abstract

CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.

Abstract

CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Biotechnology
Life Sciences > Animal Science and Zoology
Life Sciences > Agronomy and Crop Science
Life Sciences > Genetics
Language:English
Date:December 2019
Deposited On:31 Jan 2020 14:56
Last Modified:29 Jul 2020 13:15
Publisher:Springer
ISSN:0962-8819
OA Status:Hybrid
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1007/s11248-019-00168-9
PubMed ID:31482512

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