The CRISPR/Cas system was initially reported as an adaptive immune system in bacteria, consisting of three components including Cas9 nuclease and two small RNAs, CRISPR RNA (crRNA), which guides the Cas9 complex to the target sequence, and trans-activating crRNA (tracrRNA), which binds to crRNA and forms a ribonucleoprotein complex with Cas9 nuclease. The low success rates of gene cassette knock-in limit the applicability of CRISPR/Cas-mediated in vivo genome editing. ![]() In contrast, there has been only one report on the successful production of knock-in mice carrying reporter gene cassettes, essential tools for analyzing complex tissues such as brain in vivo, and the efficacy of the targeted insertion of the reporter gene was only about 10%. ![]() Ī flood of studies using CRISPR/Cas-mediated in vivo genome editing have reported the production of knockout mice and knock-in mice carrying single nucleotide substitutions combined with oligo DNA donors. The recent development of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system, a genome editing technology, has allowed for the direct manipulation of the genome in mouse zygotes in vivo ( in vivo genome editing) with extremely high efficiency, enabling the highly convenient and ultra-rapid one-step generation of genetically modified mice without embryonic stem cells. ![]() Although gene-targeted knockout and knock-in mice are invaluable tools for understanding the functions of genes in vivo, the production of such genetically modified mice has relied on gene targeting in embryonic stem cells, which is a complicated and time-consuming process.
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