Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, 157-8535, Japan.
Department of Biology, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba, 274-8510, Japan.
Sci Rep. 2017 Mar 3;7(1):59. doi: 10.1038/s41598-017-00140-9.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for creation of mutant mice with mutations mirroring those in human patients. Various methods have been developed for this purpose, including deletions, inversions, and translocations. So far, mutant mice with deletions of up to 1.2 megabases (Mb) have been generated by microinjection of the CRISPR/Cas9 system into fertilized eggs; however, a method for generation of mutant mice with a deletion of more than several Mb size is necessary because such deletions have often been identified as possible causes of human diseases. With an aim to enable the generation of disease models carrying large deletions with a breakpoint in custom-designed sequences, we developed a method for induction of an Mb-sized deletion by microinjection of a pair of sgRNAs, Cas9, and a donor plasmid into fertilized eggs. Using this method, we efficiently and rapidly generated mutant mice carrying deletions up to 5 Mb.
簇状规律间隔短回文重复 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 系统是一种用于创建具有与人类患者相似突变的突变小鼠的有用工具。为此，已经开发了各种方法，包括缺失、倒位和易位。迄今为止，通过将 CRISPR/Cas9 系统微注射到受精卵中，已经生成了长达 1.2 兆碱基 (Mb) 的缺失突变小鼠；然而，需要一种生成具有超过几个 Mb 大小缺失的突变小鼠的方法，因为这种缺失通常被认为是人类疾病的可能原因。为了能够生成具有在定制设计序列中具有断点的大缺失的疾病模型，我们开发了一种通过将一对 sgRNA、Cas9 和供体质粒微注射到受精卵中来诱导 Mb 大小缺失的方法。使用这种方法，我们高效快速地生成了携带长达 5 Mb 缺失的突变小鼠。
