AsianScientist (May 6, 2015) – Scientists have developed a cloning-free, highly efficient gene knock-in method using a modified version of the CRISPR-Cas9 system. Their results have been published in Genome Biology.
Genome editing using the clustered, regularly interspaced, short palindromic repeat (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, Tokyo Medical & Dental University (TMDU) have now overcome this issue by developing a highly efficient CRISPR-Cas9 system, which enables the targeted insertion of long gene cassette including enhanced green fluorescent protein (EGFP) into mouse genome in fertilized eggs, with an efficiency of up to approximately 50 percent.
The team reproduced the natural state of CRISPR/Cas system, which consists of three components: Cas9 protein, CRISPR ribonucleic acid (crRNA) and trans activating crRNA, instead of commonly used two-component system which consists of Cas9 messenger RNA (mRNA) and single guide RNA (sgRNA). This leads to significantly high efficiency. The improved CRISPR/Cas system further provides highly convenient and accurate gene modification and its successful transmission to the next generations.
This improved CRISPR-Cas9 system has potential for a variety of applications, including the 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 the production of livestock, fishes, plants and microorganisms carrying useful traits.
The article can be found at: Aida et al. (2015) Cloning-free CRISPR/Cas System Facilitates Functional Cassette Knock-in In Mice.
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Source: Tokyo Medical and Dental University.
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