A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells
A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells
复制标题
用于哺乳动物细胞基因表达多重激活的 microRNA 门控 thgRNA 平台
DOI：
10.1039/d2cc01478e
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发表时间：
2022
影响因子：
4.9
通讯作者：
Chen, Wilfred
中科院分区：
文献类型：
--
作者：
Hunt, Victoria M.;Chen, Wilfred
为了有效地将细胞调控网络重编程为期望的表型，关键是具有以时空方式提供精确基因调控的能力。我们之前已经工程化了支点门控引导RNA（thgRNA），以使用合成的RNA触发物在哺乳动物细胞中实现dCas9介导的转录上调的条件激活。在这里，我们证明了microRNA（miR）门控的thgRNA可以被II型RNA聚合酶转录，以允许使用mRNA和miR的多重转录激活。只有通过miR介导的侧翼5′帽和3′ poly A尾的适当加工以及mRNA通过链置换解除发夹阻断才能实现激活。这种新的与门设计被利用来引发基于特定泛素的诱导表达的条件性蛋白质降解。这种新的策略可能会以RNA响应的方式找到许多新的应用。
To effectively reprogram cellular regulatory networks towards desired phenotypes, it is critical to have the ability to provide precise gene regulation in a spatiotemporal manner. We have previously engineered toehold-gated guide RNA (thgRNA) to enable conditional activation of dCas9-mediated transcriptional upregulation in mammalian cells using synthetic RNA triggers. Here, we demonstrate that microRNA (miR)-gated thgRNAs can be transcribed by type II RNA polymerase to allow multiplexed transcriptional activation using both mRNA and miR. Activation is achieved only by proper miR-mediated processing of the flanking 5′ cap and 3′ poly A tail and hairpin unblocking by mRNA via strand displacement. This new AND-gate design is exploited to elicit conditional protein degradation based on induced expression of a specific ubiquibody. This new strategy may find many new applications in an RNA-responsive manner.