High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage.,Cell Reports

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High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage.
Cell Reports ( IF 7.5 ) Pub Date : 2019-03-26 , DOI: 10.1016/j.celrep.2019.02.094
Ian M Slaymaker ₁ , Pablo Mesa ₂ , Max J Kellner ₃ , Soumya Kannan ₄ , Edward Brignole ₅ , Jeremy Koob ₆ , Patricia R Feliciano ₅ , Stefano Stella ₂ , Omar O Abudayyeh ₄ , Jonathan S Gootenberg ₇ , Jonathan Strecker ₁ , Guillermo Montoya ₂ , Feng Zhang ₁

Affiliation

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Structural Molecular Biology Group, University of Copenhagen, Copenhagen, Denmark.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department for Biochemistry and Cell Biology, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard University, Boston, MA 02115, USA.

Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells.

中文翻译：

Cas13b 的高分辨率结构以及 RNA 靶向和切割的生化表征。

VI 型 CRISPR-Cas 系统包含可编程单效应器 RNA 引导的 RNase，包括 Cas13b（四个已知家族成员之一）。 Cas13b已用于RNA编辑和核酸检测，其线性结构域架构和CRISPR RNA (crRNA)结构在VI型CRISPR效应子中是独一无二的。在这里，我们以 1.65 Å 的分辨率报道了与 crRNA 结合的普雷沃氏菌 Cas13b (PbuCas13b) 的晶体结构。该结构与分析 Cas13b 稳定性、动力学和功能的生化实验相结合，为 Cas13b 靶标 RNA 识别提供了机制模型，并识别了负责靶标和切割特异性的特征。基于这些观察结果，我们生成了具有改变的切割偏好的 Cas13b 变体，这可能会扩展基于核酸酶的 RNA 检测分析的效用以及 Cas13b 在哺乳动物细胞中的其他应用。

更新日期：2019-03-27

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