Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity

Mol Cell. 2019 Jan 17;73(2):264-277.e5. doi: 10.1016/j.molcel.2018.11.007. Epub 2018 Nov 29.


Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus CsmcrRNA binary, CsmcrRNA-target RNA and CsmcrRNA-target RNAanti-tag ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of CsmcrRNA adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into CsmcrRNA complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.

Keywords: 5′ repeat tag pairing controls autoimmunity; DNase activity regulation by Glu residues; RNA cleavage with 6-nt periodicity; T. onnurineus Csm; cryo-EM; ternary complex assembly with crRNA and target RNA; type III-A CRISPR-Cas complexes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoimmunity*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / immunology
  • Bacterial Proteins / metabolism*
  • Bacterial Proteins / ultrastructure
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / immunology
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Associated Proteins / ultrastructure
  • CRISPR-Cas Systems* / genetics
  • CRISPR-Cas Systems* / immunology
  • Clustered Regularly Interspaced Short Palindromic Repeats* / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats* / immunology
  • Cryoelectron Microscopy
  • Deoxyribonucleases / genetics
  • Deoxyribonucleases / immunology
  • Deoxyribonucleases / metabolism*
  • Deoxyribonucleases / ultrastructure
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Escherichia coli / immunology
  • Gene Expression Regulation, Bacterial
  • Models, Molecular
  • Multiprotein Complexes
  • Mutation
  • Nucleic Acid Conformation
  • Protein Conformation
  • RNA Stability*
  • RNA, Bacterial / genetics
  • RNA, Bacterial / immunology
  • RNA, Bacterial / metabolism*
  • RNA, Bacterial / ultrastructure
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / immunology
  • RNA-Binding Proteins / metabolism*
  • RNA-Binding Proteins / ultrastructure
  • Structure-Activity Relationship
  • Thermococcus / enzymology
  • Thermococcus / genetics
  • Thermococcus / immunology


  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Multiprotein Complexes
  • RNA, Bacterial
  • RNA-Binding Proteins
  • Deoxyribonucleases