Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions.
Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions.
复制标题
有条件引导 RNA 通过两个中间发夹，实现可编程 CRISPR/Cas9 功能：建立内源 RNA 表达之间的调控连接。
DOI：
10.1093/nar/gkaa842
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发表时间：
2020-11-18
影响因子：
14.9
通讯作者：
Xu L
中科院分区：
文献类型：
--
作者：
Lin J;Liu Y;Lai P;Ye H;Xu L
为核酸计算而开发的各种纳米器件为构建用于操纵基因表达的通用合成电路提供了巨大的机会。在我们的研究中，我们利用双发夹介导的核酸链位移作为条件引导RNA的加工接头，旨在通过可编程的CRISPR/Cas9功能在自然发生的RNA表达之间建立人工连接。这种双发夹接头具有序列转换机制，其中随机触发链可以加工释放不受约束的序列无关链，从而激活自抑制引导RNA进行条件基因调控。该中间处理器以荧光报告系统为特征，用于调控CRISPR/Cas9的结合活性。利用质粒在原位产生这种序列转换机制，我们在大肠杆菌和人类细胞中实现了由其他不相关的内源性RNA控制的内源性RNA表达的自主遗传调控。与先前报道的链位移遗传回路不同，这种先进的核酸纳米机器提供了一种新的方法，可以在自然发生的内源性rna之间建立调节连接。除了CRISPR系统，我们期望这种双发夹机器可以作为一个通用的处理接头，广泛应用于其他基于rna的遗传电路的开发。
A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.
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