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 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.
Highlights
Adaptive immune systems consisting of CRISPR and CRISPR-associated (Cas) genes defend prokaryotes from foreign pathogens
In a CRISPR-Cas system, Cas proteins form a complex with a small CRISPR RNA to detect and cleave invading nucleic acid (DNA or RNA) that has a complementary sequence to the crRNA.[1]
The results showed that the Csm complex displays a spiral-like architecture, with two spirally stacked density columns, including a single Cas[10], a single Csm[4], and multiple copies of Csm[2] and Csm[3]
Summary
Adaptive immune systems consisting of CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) genes defend prokaryotes from foreign pathogens. Adaptive immune systems consisting of CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-. In a CRISPR-Cas system, Cas proteins form a complex with a small CRISPR RNA (crRNA) to detect and cleave invading nucleic acid (DNA or RNA) that has a complementary sequence to the crRNA.[1] CRISPR-Cas systems fall into 2 classes, multi-Cas types I, III and IV (Class 1), and single-Cas types II, V and VI (Class 2). The type III-A CRISPR-Csm complex contains five Cas proteins (Cas[10], and Csm2–5) and a crRNA, with the predicted stoichiometry of Cas101:Csm23:Csm35:Csm41:Csm51:crRNA1 (containing a 40 nt crRNA) for Csm-40, and Cas101:Csm26:Csm310:Csm41:Csm[51]: crRNA1 (containing a 72 nt crRNA) for Csm-72, respectively.[2] The Csm complex, with the RNA cleavage site located in
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