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首页> 美国卫生研究院文献>Nucleic Acids Research >Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions
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Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions

机译：条件导向RNA通过两种中间发夹进行可编程CRISPR / CAS9功能：在内源性RNA表达式之间建立调节性连接

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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.
机译：为核酸计算开发的各种纳米切口提供了构建用于操纵基因表达的多功能合成电路的机会。在我们的研究中，通过使用双发夹介导的核酸链位移作为条件引导RNA的加工接头，我们的目的是通过可编程CRISPR / CAS9功能在天然存在的RNA表达之间构建人工连接。该双发夹接头具有序列切换机械，其中可以将随机触发股线加工以释放无约束的序列依赖性链，从而激活自我抑制导向RNA以进行条件基因调控。该中间处理器的特征在于荧光报告系统，并施用用于调节CRISPR / CAS9结合活性。使用质粒原位产生该序列切换机械，我们实现了由其他无关的内源RNA控制的内源性RNA表达的自主遗传调节，在大肠杆菌和人体细胞中。与先前报道的股线遗传遗传电路不同，这种先进的核酸纳米载荷提供了一种新的方法，可以在天然存在的内源性RNA之间建立调节性关系。除了CRISPR系统之外，我们预计这款双发夹机可以作为广泛应用的一般加工接头，以便在其他基于RNA的遗传电路的发展中。

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