In vitro assembly of thermostable Csm complex in CRISPR–Cas type III/A system

Abstracts The CRISPR–Cas system is the prokaryotic immune response that destroys invading foreign nucleic acids. Based on the architecture and distinct mechanism of targeting, the CRISPR–Cas system is classified into six types (I–VI). The Csm complex belongs to the type III system and consists of five subunits (Cas10 and Csm2–5) and a crRNA. The Csm complex targets RNA and RNA-dependent single-strand DNA. Here, we present a protocol for in vitro reconstitution of a Csm complex from a hyperthermophilic archaeon Thermococcus onnurineus NA1 ( To Csm complex). The method consists of coexpression and copurification of the subunits, in vitro synthesis of the crRNA and assembly of the To Csm complex. Purification with heat treatment and affinity and size exclusion chromatography resulted in homogeneous Cas10/Csm4 and Csm2/Csm5 binary complexes, while in vitro transcription with the T7 promoter enabled synthesis of the crRNA. Addition of each component in the presence of the crRNA with a molar ratio of Cas10/Csm4:Csm3:Csm2/Csm5:crRNA = 1:3:2:1 yielded an assembled functional Csm complex. This protocol for reconstitution of the Csm complex is presumably applicable to other thermostable effector complexes, which would allow biochemical, structural, or functional studies of the CRISPR–Cas type III/A system in vitro.