Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice.
Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice.
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DOI：
10.1016/j.fob.2014.06.007
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发表时间：
2014
期刊：
影响因子：
2.6
通讯作者：
Lloyd, Kevin C. Kent
中科院分区：
文献类型：
--
作者：
Lee, Angus Yiu-fai;Lloyd, Kevin C. Kent
我们通过在小鼠中使用配对的Cas9 n（切口酶）、gRNA和单个DNA模板产生了floxed等位基因。我们证实了floxed等位基因是生殖系传递的，并在F1后代中发挥作用。在C57 BL/6 N背景小鼠中产生含类异戊二烯合酶结构域（Ispd）基因的floxed等位基因。这种方法可用于产生潜在胚胎致死基因的敲除小鼠。CRISPR/Cas9技术是一种非常有前途的小鼠基因组编辑工具，可能克服胚胎干细胞（ES）中更传统的基因靶向方法所需的成本和时间。最近，与野生型核酸酶相比，已发现配对Cas9切口酶（Cas9 n）与单向导RNA（sgRNA）分子组合增强基因组编辑的特异性，同时减少脱靶效应。配对的Cas9 n已被证明与Cas9一样有效，用于通过基因组中的非同源末端连接和靶向缺失产生插入和缺失（indel）突变。然而，插入关键外显子侧翼的loxP位点以产生靶基因的条件等位基因的有效且可靠的方法仍然是难以实现的目标。在这项研究中，我们将Cas9 n RNA与sgRNA以及编码两个loxP位点的单个DNA模板一起显微注射到C57 BL/6 NCr单细胞期合子的原核和细胞质中，所述两个loxP位点位于含有类异戊二烯合酶的结构域（Ispd）的外显子2的侧翼（floxing）。手术转移后，产生了一只表达条件等位基因的F0小鼠（出生活仔的频率为1.8%）。floxed等位基因通过生殖系传递到F1后代，并且可以使用Cre重组酶成功重组。这项研究表明，使用配对的Cas9 n和单个DNA模板可以有效地实现条件靶向。
We generated a floxed allele by using paired Cas9n (nickase), gRNAs and single DNA template in mouse. We confirmed that the floxed allele was germline transmitted and functional in F1 offspring. A floxed allele of the isoprenoid synthase containing domain (Ispd) gene in C57BL/6N background mice was created. This method can be used to generate knockout mice for genes that are potentially embryonic lethal. CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.
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影响因子：
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作者：
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通讯作者：
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作者：
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影响因子：
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作者：
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影响因子：
46.9
作者：
通讯作者：
--
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