Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells.
Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells.
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DOI：
10.1016/j.bbrc.2016.11.129
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发表时间：
2017-01-22
影响因子：
3.1
通讯作者：
Mao Y
中科院分区：
文献类型：
--
作者：
Dong F;Xie K;Chen Y;Yang Y;Mao Y
CRISPR/Cas9已广泛用于许多生物体的基因组编辑。许多人类疾病是由多重突变引起的。 CRISPR/Cas9 系统提供了一种在基因组中引入多种突变的潜在工具。为了模拟人类疾病中复杂的基因组变异，例如多基因缺失或突变，需要同时引入两个或多个小引导RNA (sgRNA)。这可以通过构建体中单独的 Pol III 启动子来实现。然而，有限的酶位点和增加的插入大小降低了构建体的效率。在这里，我们报告了一种使用多顺反子-tRNA-gRNA (PTG) 策略在一个构建体中快速组装多个 sgRNA 的策略。利用哺乳动物细胞中的内源性 tRNA 加工系统，我们仅使用一个 Pol III 启动子即可高效表达多种 sgRNA。使用携带 PTG 的一体化构建体，我们同时破坏人类细胞中多种组蛋白脱乙酰酶 (HDAC) 的脱乙酰酶结构域。我们证明多个 HDAC 缺失显着影响 Wnt 信号通路的激活。因此，该方法能够有效地靶向多个基因，并为建立模仿人类疾病的突变细胞提供有用的工具。
CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct. Here, we report a strategy to quickly assembly multiple sgRNAs in one construct using a polycistronic-tRNA-gRNA (PTG) strategy. Taking advantage of the endogenous tRNA processing system in mammalian cells, we efficiently express multiple sgRNAs driven using only one Pol III promoter. Using an all-in-one construct carrying PTG, we disrupt the deacetylase domain in multiple histone deacetylases (HDACs) in human cells simultaneously. We demonstrate that multiple HDAC deletions significantly affect the activation of the Wnt-signaling pathway. Thus, this method enables to efficiently target multiple genes and provide a useful tool to establish mutated cells mimicking human diseases.