Generation of a Double KO Mouse by Simultaneous Targeting of the Neighboring Genes Tmem176a and Tmem176b Using CRISPR/Cas9: Key Steps from Design to Genotyping

详细信息    查看全文

• 作者：Auré ; lie Lemoine^a ; Gaë ; lle Chauveau-Le Friec^b ; Francina Langa^b ; Cé ; dric Louvet^a ; ^{cedric.louvet@univ-nantes.fr" class="auth_mail" title="E-mail the corresponding author}
• 关键词：CRISPR/Cas9 ; C57BL/6 ; Double KO ; Genotyping ; NHEJ ; Deletion ; Inversion ; Off-targets ; Mosaicism
• 刊名：Journal of Genetics and Genomics
• 出版年：2016
• 出版时间：20 May 2016
• 年：2016
• 卷：43
• 期：5
• 页码：329-340
• 全文大小：1323 K

文摘

The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy. While complex genome editing in the mouse since the 1990s has been dominated by the use of embryonic stem (ES) cells, CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes. Yet, this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project. Here, we describe the key steps leading to successful generation of a double knockout (KO) mouse by simultaneously targeting two homolog genes, Tmem176a and Tmem176b, which are located in the same genomic locus. Additionally, we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain. Thus, presented as a detailed case study that should be helpful to the non-specialists, we focus on the genotyping strategy to anticipate the various possibilities.