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Highly efficient CRISPR knock-in in mouse

May 1, 2015
Tokyo Medical and Dental University
The CRISPR/Cas system, which is based on chemically synthesized small RNAs and commercially available Cas9 enzyme, has enabled long gene-cassette knock-in in mice with highest efficiency ever reported, scientists report.

Genome editing using CRISPR/Cas system has enabled direct modification of the mouse genome in fertilized mouse eggs, leading to rapid, convenient, and efficient one-step production of knockout mice without embryonic stem cells. In contrast to the ease of targeted gene deletion, the complementary application, called targeted gene cassette insertion or knock-in, in fertilized mouse eggs by CRISPR/Cas mediated genome editing still remains a tough challenge.

Professor Kohichi Tanaka and Dr. Tomomi Aida at Laboratory of Molecular Neuroscience, Medical Research Institute, TMDU has now overcome this issue by developing innovative highly efficient CRISPR/Cas system, which resulted in targeted insertion of long gene cassette including enhanced green fluorescent protein (EGFP) into mouse genome in fertilized eggs with efficiency up to approx. 50%. The team reproduced the natural state of CRISPR/Cas system, which consists of three components: Cas9 protein, CRISPR RNA (crRNA), and trans activating crRNA (crRNA), instead of commonly used two-component system which consists of Cas9 mRNA and single guide RNA (sgRNA), leading to extremely high efficiency. The improved CRISPR/Cas system further provides highly convenient and accurate gene modification, and its successful transmission to the next generations.

The new work was published in the open access journal Genome Biology as an article entitled "Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice" on April 29, 2015.

This improved CRISPR/Cas system will be useful for a variety of applications, including creation of humanized mice for modeling of genetic diseases, drug metabolisms, immunity, and infectious diseases. Further, accurate targeted insertion will improve the safety of gene therapy in human patients in the future. The new system can be also applied to other purposes such as production of livestock, fishes, plants, and microorganisms carrying useful traits.

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Materials provided by Tokyo Medical and Dental University. Note: Content may be edited for style and length.

Journal Reference:

  1. Tomomi Aida, Keiho Chiyo, Takako Usami, Harumi Ishikubo, Risa Imahashi, Yusaku Wada, Kenji F Tanaka, Tetsushi Sakuma, Takashi Yamamoto, Kohichi Tanaka. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biology, 2015; 16 (1) DOI: 10.1186/s13059-015-0653-x

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Tokyo Medical and Dental University. "Highly efficient CRISPR knock-in in mouse." ScienceDaily. ScienceDaily, 1 May 2015. <>.
Tokyo Medical and Dental University. (2015, May 1). Highly efficient CRISPR knock-in in mouse. ScienceDaily. Retrieved November 28, 2023 from
Tokyo Medical and Dental University. "Highly efficient CRISPR knock-in in mouse." ScienceDaily. (accessed November 28, 2023).

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