CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo.
CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo.
复制标题
DOI：
10.1038/srep22312
复制
发表时间：
2016-02-29
影响因子：
4.6
通讯作者：
Xi Y
中科院分区：
文献类型：
--
作者：
Chang H;Yi B;Ma R;Zhang X;Zhao H;Xi Y
MicroRNA是一种小的非编码RNA分子，在转录后/翻译水平上调控基因表达。针对microRNA功能丧失的研究，目前已经开发了许多方法，如反义抑制剂和海绵等，但这些传统方法的稳健性、特异性和稳定性都不尽如人意。CRISPR/cas9系统作为一种新型的基因组编辑工具正在生物学/医学研究中兴起，但其在microRNA研究中的适应症尚未得到专门研究。在这项研究中，我们克隆了CRISPR/cas9构建体，其具有特异性靶向选定microRNA的生物合成加工位点的单向导RNA;我们发现CRISPR/cas9可以稳健地特异性降低这些microRNA的表达高达96%。CRISPR/cas9在控制相同家族或具有高度保守序列的microRNA上的交叉脱靶效应方面也显示出独特的益处。更重要的是，我们首次在体外和体内模型中证明了CRISPR/cas9对microRNA敲低表型的长期稳定性。
MicroRNAs are small and non-coding RNA molecules with the master role in regulation of gene expression at post-transcriptional/translational levels. Many methods have been developed for microRNA loss-of-function study, such as antisense inhibitors and sponges; however, the robustness, specificity, and stability of these traditional strategies are not highly satisfied. CRISPR/cas9 system is emerging as a novel genome editing tool in biology/medicine research, but its indication in microRNA research has not been studied exclusively. In this study, we clone CRISPR/cas9 constructs with single-guide RNAs specifically targeting biogenesis processing sites of selected microRNAs; and we find that CRISPR/cas9 can robustly and specifically reduce the expression of these microRNAs up to 96%. CRISPR/cas9 also shows an exclusive benefit in control of crossing off-target effect on microRNAs in the same family or with highly conserved sequences. More significantly, for the first time, we demonstrate the long term stability of microRNA knockdown phenotype by CRISPR/cas9 in both in vitro and in vivo models.