1. Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d
- Author
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Zhang, Cheng, Konermann, Silvana, Brideau, Nicholas J, Lotfy, Peter, Wu, Xuebing, Novick, Scott J, Strutzenberg, Timothy, Griffin, Patrick R, Hsu, Patrick D, and Lyumkis, Dmitry
- Subjects
Genetics ,Bioengineering ,Biotechnology ,Underpinning research ,1.1 Normal biological development and functioning ,Quality Education ,CRISPR-Cas Systems ,Clustered Regularly Interspaced Short Palindromic Repeats ,Cryoelectron Microscopy ,Endonucleases ,HEK293 Cells ,Humans ,Molecular Conformation ,RNA ,RNA ,Guide ,Kinetoplastida ,Ribonucleases ,CRISPR ,Cas13 ,Cas13d ,Cas9 ,RNA interference ,RNA targeting ,cryo-EM ,shRNA ,structure ,type VI ,Biological Sciences ,Medical and Health Sciences ,Developmental Biology - Abstract
CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
- Published
- 2018