Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.
Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.
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DOI：
10.1093/nar/gkab100
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发表时间：
2021-03-18
影响因子：
14.9
通讯作者：
Beisel CL
中科院分区：
文献类型：
--
作者：
Collins SP;Rostain W;Liao C;Beisel CL
CRISPR技术越来越需要核酸酶活性的时空和剂量控制。一种有希望的策略涉及通过工程化引导RNA（gRNA）将核酸酶活性与细胞的转录状态联系起来，使其仅在与“触发”RNA复合后发挥功能。然而，标准gRNA开关设计不允许独立选择触发序列和指导序列，限制了gRNA开关应用。在这里，我们展示了Cas 12 a gRNA开关的模块化设计，该模块化设计使这些序列的选择更加复杂。Cas 12 a gRNA的5′端融合到两个不同且不重叠的结构域：一个碱基与gRNA重复序列配对，阻断Cas 12 a识别所需发夹的形成;另一个与RNA触发物杂交，刺激gRNA重复序列的重折叠和随后的gRNA依赖性Cas 12 a活性。使用无细胞转录-翻译系统和大肠杆菌，我们表明设计的gRNA开关可以响应不同的触发器并靶向不同的DNA序列。调节感觉结构域的长度和组成改变了gRNA开关的性能。最后，gRNA开关可以被设计成感测仅在特定生长条件下表达的内源RNA，使得Cas 12 a靶向活性依赖于细胞代谢和应激。因此，我们的设计框架进一步实现了CRISPR活动与细胞状态的联系。
CRISPR technologies increasingly require spatiotemporal and dosage control of nuclease activity. One promising strategy involves linking nuclease activity to a cell's transcriptional state by engineering guide RNAs (gRNAs) to function only after complexing with a ‘trigger’ RNA. However, standard gRNA switch designs do not allow independent selection of trigger and guide sequences, limiting gRNA switch application. Here, we demonstrate the modular design of Cas12a gRNA switches that decouples selection of these sequences. The 5′ end of the Cas12a gRNA is fused to two distinct and non-overlapping domains: one base pairs with the gRNA repeat, blocking formation of a hairpin required for Cas12a recognition; the other hybridizes to the RNA trigger, stimulating refolding of the gRNA repeat and subsequent gRNA-dependent Cas12a activity. Using a cell-free transcription-translation system and Escherichia coli, we show that designed gRNA switches can respond to different triggers and target different DNA sequences. Modulating the length and composition of the sensory domain altered gRNA switch performance. Finally, gRNA switches could be designed to sense endogenous RNAs expressed only under specific growth conditions, rendering Cas12a targeting activity dependent on cellular metabolism and stress. Our design framework thus further enables tethering of CRISPR activities to cellular states.
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影响因子：
11.3
作者：
Beisel CL;Storz G
通讯作者：
Storz G
影响因子：
64.8
作者：
Abudayyeh OO;Gootenberg JS;Essletzbichler P;Han S;Joung J;Belanto JJ;Verdine V;Cox DBT;Kellner MJ;Regev A;Lander ES;Voytas DF;Ting AY;Zhang F
通讯作者：
Zhang F
影响因子：
16
作者：
Dolan, Adam E.;Hou, Zhonggang;Zhang, Yan
通讯作者：
Zhang, Yan
影响因子：
56.9
作者：
Chen, Janice S.;Ma, Enbo;Doudna, Jennifer A.
通讯作者：
Doudna, Jennifer A.
影响因子：
46.9
作者：
Cameron, Peter;Coons, Mary M.;Sternberg, Samuel H.
通讯作者：
Sternberg, Samuel H.