Department of Biosystems Science, Institute for Frontier and Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan.
Department of Mammalian Regulatory Networks, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
Sci Rep. 2020 Aug 24;10(1):14120. doi: 10.1038/s41598-020-70804-6.
CRISPR/Cas-mediated genome editing is a powerful tool for generating genetically mutated cells and organisms. Linearisation of donor cassettes with this system has been shown to facilitate both transgene donor insertion and targeted knock-in. Here, we developed a donor plasmid that we name pCriMGET (plasmid of synthetic CRISPR coded RNA target sequence-equipped donor plasmid-mediated gene targeting), in which an off-target free synthetic CRISPR coded RNA-target sequence (syn-crRNA-TS) is incorporated with a multi-cloning site, where a donor cassette can be inserted. With co-expression of Cas9 and the syn-crRNA-TS guide RNA (gRNA), pCriMGET provides a linearised donor cassette in vivo, thereby promoting the transgene donor insertion and targeted knock-in. When co-injected with Cas9 protein and gRNA into murine zygotes, pCriMGET yielded around 20% transgene insertion in embryos. This method also achieved more than 25% in-frame knock-in at the mouse Tbx3 gene locus without predicted insertion-deletion mutations using a transgene donor with 400-bp homology arms. pCriMGET is therefore useful as a versatile CRISPR/Cas9-cleavable donor plasmid for efficient integration and targeted knock-in of exogenous DNA in mice.
CRISPR/Cas 介导的基因组编辑是一种强大的工具，可用于生成遗传突变细胞和生物体。该系统中供体盒的线性化已被证明有利于转基因供体插入和靶向敲入。在这里，我们开发了一种称为 pCriMGET（带有合成 CRISPR 编码 RNA 靶序列的供体质粒介导的基因靶向质粒）的供体质粒，其中包含无脱靶的合成 CRISPR 编码 RNA 靶序列（syn-crRNA-TS）和多克隆位点，供体盒可插入其中。在共表达 Cas9 和 syn-crRNA-TS 指导 RNA（gRNA）的情况下，pCriMGET 在体内提供线性化的供体盒，从而促进转基因供体插入和靶向敲入。当将 Cas9 蛋白和 gRNA 共注射到小鼠受精卵中时，pCriMGET 在胚胎中产生约 20%的转基因插入。该方法还使用带有 400bp 同源臂的转基因供体在小鼠 Tbx3 基因座上实现了超过 25%的框内敲入，而没有预测的插入缺失突变。因此，pCriMGET 可用作一种通用的 CRISPR/Cas9 可切割供体质粒，用于在小鼠中高效整合和靶向敲入外源 DNA。
