Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs.
Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs.
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DOI：
10.1038/ncomms14633
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发表时间：
2017-03-03
影响因子：
16.6
通讯作者：
Fulga TA
中科院分区：
文献类型：
--
作者：
Ferry QR;Lyutova R;Fulga TA
基于CRISPR的转录调节因子（CRISPR-TR）通过允许任何靶基因的特异性激活或抑制，改变了当前的合成生物学格局。在这里，我们报告了一个模块化和通用的框架，能够在哺乳动物细胞中快速实施诱导型CRISPR-TR。这种策略依赖于在单向导RNA（sgRNA）的5′端设计间隔子阻断发夹（SBH）结构，其废除了CRISPR转录激活因子的功能。通过用配体控制的RNA切割单位替换SBH环，我们证明了对被编程为响应遗传编码或外部递送的触发物的静止sgRNA的条件激活。我们使用这个系统耦合多个合成和内源性靶基因与特定的诱导剂，组装基因调控模块，展示平行和正交的转录程序。我们预计，这种“即插即用”的方法将是合成生物学工具包的一个有价值的补充，促进对天然基因电路的理解和基于细胞的治疗策略的设计。能够以可控的方式调节基因表达是合成生物学的承诺之一，CRISPR-Cas9是为实现这一目标而开发的工具之一。在这里，作者使用工程化的指导RNA开发了一种诱导型CRISPR平台。
CRISPR-based transcription regulators (CRISPR-TRs) have transformed the current synthetic biology landscape by allowing specific activation or repression of any target gene. Here we report a modular and versatile framework enabling rapid implementation of inducible CRISPR-TRs in mammalian cells. This strategy relies on the design of a spacer-blocking hairpin (SBH) structure at the 5′ end of the single guide RNA (sgRNA), which abrogates the function of CRISPR-transcriptional activators. By replacing the SBH loop with ligand-controlled RNA-cleaving units, we demonstrate conditional activation of quiescent sgRNAs programmed to respond to genetically encoded or externally delivered triggers. We use this system to couple multiple synthetic and endogenous target genes with specific inducers, and assemble gene regulatory modules demonstrating parallel and orthogonal transcriptional programs. We anticipate that this ‘plug and play' approach will be a valuable addition to the synthetic biology toolkit, facilitating the understanding of natural gene circuits and the design of cell-based therapeutic strategies. Being able to regulate gene expression in a controllable manner is one of the promises of synthetic biology, and CRISPR-Cas9 is one of the tools being developed to achieve that goal. Here the authors develop an inducible CRISPR platform using an engineered guide RNA.