Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry

Cell Research
Minghui GuoZhiwei Huang

Abstract

The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and bioche...Continue Reading

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Citations

Nov 19, 2019·Frontiers in Microbiology·Do-Heon GuJeong-Sun Kim
Mar 15, 2020·Proceedings of the National Academy of Sciences of the United States of America·Kaiming ZhangWah Chiu
May 21, 2020·FEMS Microbiology Reviews·Avery Roberts, Rodolphe Barrangou
Jul 28, 2020·Current Opinion in Structural Biology·Rafael MolinaGuillermo Montoya
Nov 24, 2020·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·Mingtao ZhangXiao Zhu
Jun 6, 2021·Nature Reviews. Molecular Cell Biology·Ning Jia, Dinshaw J Patel
Sep 14, 2021·Chemical Physics·Yamini Thakur, Rama Pande

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Methods Mentioned

BETA
cleavage assay
ion
size-exclusion chromatography
electrophoresis
PCR
fluorescence imaging

Software Mentioned

CTFFIND4
PSIPRED
CHIMERA
EMAN2
CryoSparc
MotionCor2
RELION
space
MolProbity
COOT

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