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Abstract：
The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging1-6. Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA1,7. Though Cas13 is not activated by double-stranded RNA (dsRNA) in vitro, it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured2,8. Understanding Cas13\'s mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown9. Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3\' to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection, enhancing mismatch discrimination by up to 50-fold and enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.
摘要：
RNA靶向CRISPR核酸酶Cas13已成为从核酸检测到转录组工程和RNA成像1-6的应用的强大工具。Cas13通过CRISPRRNA(crRNA)与靶RNA中的互补单链RNA(ssRNA)前间隔子的杂交而被激活1,7。虽然Cas13在体外不被双链RNA（dsRNA）激活，矛盾的是，它在绝大多数RNA高度结构化的环境中证明了强大的RNA靶向2,8。了解Cas13的结合和激活机制将是提高其检测和干扰RNA能力的关键；然而，Cas13结合结构化RNA的机制仍然未知9.这里,我们使用大规模多重筛选系统探测LwaCas13a响应RNA结构扰动的激活机制。我们发现，二级结构有两种不同的序列无关模式影响Cas13活性：前间隔区的结构与crRNA竞争，可以通过链置换机制被破坏，而在区域3\'到原型间隔区的结构具有变构抑制作用。我们利用链置换过程的动力学性质来改进基于Cas13的RNA检测，将错配辨别能力提高多达50倍，并能够在低(<1%)等位基因频率下鉴定序列不可知的突变。我们的工作为基于CRISPR的核酸检测树立了新的标准，并将实现智能和二级结构指导的靶标选择，同时还扩大了可用于Cas13靶向的RNA范围。
