Structure-based design of gRNA for Cas13
Structure-based design of gRNA for Cas13
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DOI：
10.1038/s41598-020-68459-4
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发表时间：
2020-07-14
影响因子：
4.6
通讯作者：
Ito, Tatsuo
中科院分区：
文献类型：
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作者：
Bandaru, Srinivas;Tsuji, Mika Higashide;Ito, Tatsuo
Cas13内切酶的活性取决于RNA局部二级结构，对单链（SS）区域有强烈的偏好。因此，鉴定SS区域以进行有效的Cas13介导的RNA敲低变得必不可少。本文将实验结构序列数据与预测结构模型相结合，提出合理的gRNA设计。利用XIST转录物的结构序列数据，我们观察到靶向SS区的grna比靶向双链（DS）区的grna更能诱导转录物的敲低和切割。此外，我们在gRNA中发现了“中心种子区”，当靶向SS区域时，它有效地促进了Cas13介导的切割。在接下来的研究中，我们考虑了无法获得实验结构-序列数据的情况，因此我们使用滑膜肉瘤中显示的SS18-SSX2融合转录物并计算预测其结构。我们观察到，与靶向DS区域的grna相比，从结构上预测靶向SS区域的grna更有效地诱导了坏死。综上所述，为了实现有效的RNA敲低，本文提出的Cas13介导的靶向策略强调利用结构信息设计特异性靶向SS区域的grna。此外，该策略反过来可以预期缩小gRNA设计的搜索空间（通过专门针对SS区域），特别是当lncrna是目标时。
Cas13 endonuclease activity depends on the RNA local secondary structure with strong preference for single-stranded (SS) regions. Hence, it becomes indispensable to identify the SS regions for effective Cas13 mediated RNA knockdown. We herein present rational gRNA design by integrating experimental structure-seq data and predicted structural models. Utilizing structure-seq data for XIST transcript, we observed that gRNAs targeting the SS regions significantly induce transcript knockdown and cleavage than those targeting double-stranded (DS) regions. Further, we identified the "central seed region" in the gRNA that upon targeting the SS regions efficiently facilitates Cas13 mediated cleavage. In our following pursuits, we considered the scenario wherein experimental structure-seq data is not available, hence we used SS18-SSX2 fusion transcript indicated in synovial sarcomas and computationally predicted its structure. We observed that gRNAs targeting the SS regions predicted from the structure, efficiently induced necrosis compared to gRNAs that target the DS regions. In conclusion, for the effective RNA knockdown, the Cas13 mediated targeting strategy presented herein emphasizes the designing of gRNAs specifically targeting SS regions by utilizing structural information. Further, this strategy, in turn, can be anticipated to narrow the search space for gRNA design (by exclusively targeting SS regions) especially when lncRNAs are the targets.