Genome editing with the donor plasmid equipped with synthetic crRNA-target sequence
Genome editing with the donor plasmid equipped with synthetic crRNA-target sequence
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DOI：
10.1038/s41598-020-70804-6
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发表时间：
2020-08-24
影响因子：
4.6
通讯作者：
Toyoshima, Fumiko
中科院分区：
文献类型：
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作者：
Ishibashi, Riki;Abe, Kota;Toyoshima, Fumiko
CRISPR/ cas介导的基因组编辑是产生基因突变细胞和生物体的强大工具。该系统的供体盒线性化已被证明可促进转基因供体插入和靶向敲入。在这里，我们开发了一种供体质粒，我们将其命名为pCriMGET（合成CRISPR编码RNA靶序列装备的供体质粒介导的基因靶向质粒），其中脱靶的合成CRISPR编码RNA靶序列（syn-crRNA-TS）与一个多克隆位点结合，在那里可以插入供体卡带。通过Cas9和syncrrna - ts向导RNA （gRNA）的共表达，pCriMGET在体内提供了一个线性化的供体盒，从而促进转基因供体插入和靶向敲入。当将Cas9蛋白和gRNA共同注射到小鼠受精卵中时，pCriMGET在胚胎中的转基因插入率约为20%。使用400 bp同源臂的转基因供体，该方法在小鼠Tbx3基因位点实现了超过25%的框内敲入，没有预测的插入-删除突变。因此，pCriMGET是一种多功能的CRISPR/ cas9可切割的供体质粒，可用于小鼠外源DNA的高效整合和靶向敲入。
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.