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

Journal Article · · Cell Research
 [1];  [2];  [1];  [2];  [1];  [2];  [3];  [1];  [4];  [1]
  1. Harbin Inst. of Technology (China). HIT Center for Life Sciences, School of Life Science and Technology
  2. Stanford Univ., CA (United States). Dept. of Bioengineering, and of Microbiology and Immunology, and James H. Clark Center
  3. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL). CryoEM and Bioimaging Division
  4. Stanford Univ., CA (United States). Dept. of Bioengineering, and of Microbiology and Immunology, and James H. Clark Center; SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL). CryoEM and Bioimaging Division
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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 biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5’-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3’ flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.

Research Organization:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Organization:
USDOE; National Natural Science Foundation of China (NSFC); National Institutes of Health (NIH)
Grant/Contract Number:
AC02-76SF00515; 31825008; 31422014; P41GM103832; U54GM103297; R01GM079429; S10OD021600
OSTI ID:
1528831
Journal Information:
Cell Research, Vol. 29, Issue 4; ISSN 1001-0602
Publisher:
Shanghai Institutes for Biological SciencesCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 24 works
Citation information provided by
Web of Science

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Cited By (2)

A CRISPR RNA Is Closely Related With the Size of the Cascade Nucleoprotein Complex journal October 2019
Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase journal May 2020

Figures / Tables (5)

Figure 1(p. 2)figure Figure 1
Figure 2(p. 3)figure Figure 2
Figure 3(p. 4)figure Figure 3
Figure 4(p. 5)figure Figure 4
Figure 5(p. 6)figure Figure 5

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