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Genome-Editing Technology in CRISPR/Cas System: How to Increase Knock-In Efficiency in Mouse Zygotes

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Genome Editing

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Abstract

The use of clustered regularly interspaced short palindromic repeats (CRISPR) and RNA-guided Cas9 nucleases, known as the CRISPR/Cas system, represents a major technological advance in mammalian gene disruption. CRISPR/Cas enables genome editing by inducing targeted DNA double-strand breaks (DSBs) that are repaired by error-prone, nonhomologous end-joining (NHEJ), or homology-directed repair (HDR). This system has emerged as an effective tool for gene knockout via NHEJ; however, it remains inefficient for precise editing of genome sequences depending on HDR. Nevertheless, HDR-mediated gene editing is essential for conditional knockout, introduction of reporter genes, and precise point mutation in mice. Many studies have examined, for example, conditions of Cas9 and guide RNA (gRNA), methods of their introduction, and molecules to increase efficiency. In this review, we describe various methods for increasing the efficiency of editing in mouse zygotes.

Author Contributions

T. Horii and I. Hatada wrote the manuscript.

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Abbreviations

amiRNA:

Artificial microRNA

CRISPR:

Clustered regularly interspaced short palindromic repeats

crRNA:

CRISPR RNA

DSB:

Double-strand breaks

dsDNA:

Double-stranded DNA

ESC:

Embryonic stem cell

gRNA:

Guide RNA

HDR:

Homology-directed repair

indels:

Insertions or deletions

iPSC:

Induced pluripotent stem cells

NHEJ:

Nonhomologous end-joining

ssODN:

Single-stranded oligo-DNA

TALEN:

Transcription activator-like effector nucleases

tracrRNA:

Trans-activating crRNA

ZFN:

Zinc-finger nucleases

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Acknowledgments

This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Ministry of Health, Labor, and Welfare of Japan; the National Institute of Biomedical Innovation; and the Takeda Science Foundation.

Conflicts of Interest

The authors declare no conflicts of interest.

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Authors and Affiliations

  1. Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan

    Takuro Horii & Izuho Hatada

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  1. Takuro Horii
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  2. Izuho Hatada
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Correspondence to Izuho Hatada .

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Editors and Affiliations

  1. Ottawa, Ontario, Canada

    Kursad Turksen

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Horii, T., Hatada, I. (2016). Genome-Editing Technology in CRISPR/Cas System: How to Increase Knock-In Efficiency in Mouse Zygotes. In: Turksen, K. (eds) Genome Editing. Springer, Cham. https://doi.org/10.1007/978-3-319-34148-4_5

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