Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System

Highlights

• Cryo-EM structures of three different assemblies of the Cas1, Cas2, and Csn2 proteins

• One form, with the stoichiometry Cas1₈-Cas2₄-Csn2₈, contains ∼30 bp duplex DNA

• Another comprises two of these units with an additional Cas1₈-Cas2₄ interface

• Filaments comprise Cas1₈-Cas2₄-Csn2₈ units around a continuous DNA duplex

Summary

CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas1₈-Cas2₄-Csn2₈, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas1₈-Cas2₄-Csn2₈ units (typically 2–6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.