A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.
A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.
复制标题
一种方便的基于 Cas9 的条件敲除策略，用于同时靶向小鼠中的多个基因。
DOI：
10.1038/s41598-017-00654-2
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发表时间：
2017-03-31
影响因子：
4.6
通讯作者：
Shi YS
中科院分区：
文献类型：
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作者：
Chen J;Du Y;He X;Huang X;Shi YS
探测蛋白质功能最有效的方法是描述其缺失的后果。与传统的基因敲除（KO）相比，条件敲除（cKO）提供了一种先进的基因靶向策略，可以在空间和时间限制的方式下进行基因删除。然而，对于大多数两倍体物种，广泛使用的Cre-flox条件KO （cKO）系统需要将两个等位基因中的靶向位点都置于loxP侧翼，这在实践中需要耗费时间和人力进行育种。这在处理多个基因时是相当重要的。CRISPR/Cas9基因组调控系统具有同时靶向多个位点的优势。在这里，我们提出了一种策略，可以在Cre重组酶依赖Cas9表达的小鼠中实现多基因的条件KO。通过转基因构建loxP-stop-loxP （LSL）控制的Cas9 （LSL-Cas9）和靶向EGFP的sgRNAs，我们发现荧光分子可以以cre依赖的方式被消除。我们进一步验证了这种新策略的有效性，通过在巨噬细胞中特异性地同时删除c-Maf和MafB来靶向多个位点。与传统的Cre-flox cKO策略相比，该sgRNAs-LSL-Cas9 cKO系统更简单，速度更快，使多基因的条件操作成为可能。
The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.