Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.
Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.
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DOI：
10.1038/srep05396
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发表时间：
2014-06-23
影响因子：
4.6
通讯作者：
Takada S
中科院分区：
文献类型：
--
作者：
Inui M;Miyado M;Igarashi M;Tamano M;Kubo A;Yamashita S;Asahara H;Fukami M;Takada S
引入点突变是用于证明特定核苷酸或氨基酸在遗传元件或蛋白质中的作用的基本方法，并且广泛用于基于培养的细胞和外源提供的DNA的体外实验。然而，通过修饰基因组基因座的这种方法的体内应用并不常见，部分原因是其技术和时间要求。这使得许多体外发现在体内条件下未经验证。我们在本文中应用CRISPR/Cas9系统来产生在其基因组中具有点突变的小鼠，这导致感兴趣的蛋白质中的单个氨基酸取代。通过将gRNA、hCas 9 mRNA和单链供体寡核苷酸（ssODN）显微注射到小鼠受精卵中，我们以低成本和短时间在其基因组中引入了定义的基因组修饰。单gRNA/WT hCas 9和双切口设置都是有效的。我们还发现修饰位点和gRNA靶位点之间的距离是影响取代效率的重要参数。我们相信，这是一种强大的技术，可用于检查体外结果的相关性，以及在体内系统中遗传性疾病患者中发现的突变。
Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. We herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, we introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. We also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. We believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.
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DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM
影响因子：
4.6
作者：
Kato, Tomoko;Miyata, Kohei;Sonobe, Miku;Yamashita, Satoshi;Tamano, Moe;Miura, Kento;Kanai, Yoshiakira;Miyamoto, Shingo;Sakuma, Tetsushi;Yamamoto, Takashi;Inui, Masafumi;Kikusui, Takefumi;Asahara, Hiroshi;Takada, Shuji
通讯作者：
Takada, Shuji
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
5.3
作者：
Elliott, B;Richardson, C;Jasin, M
通讯作者：
Jasin, M
影响因子：
64.5
作者：
Wang H;Yang H;Shivalila CS;Dawlaty MM;Cheng AW;Zhang F;Jaenisch R
通讯作者：
Jaenisch R