Figure 1.

Cas1, Cas2, Csn2, and Cas9 Proteins of CRISPR3-Cas System from S. thermophilus DGCC7710 Form a Stable Complex

(A) Schematic representation of the CRISPR3-Cas locus.

(B) CRISPR3-Cas system deletion mutants used to screen the complex assembly.

(C) Results from purification of the complexes using the deletion mutants of the CRISPR3-Cas system depicted in (B). The CRISPR3-Cas system variants were co-expressed with Cas1 protein bearing a StrepII tag. The complexes were purified using StrepTrap column followed by size-exclusion chromatography and analyzed on SDS-PAGE. Cas1 and Cas2 form the core complex. Csn2 binds to Cas1-Cas2 complex (or vice versa), allowing binding of Cas9.

(D) Cas1, Cas2, Csn2, and Cas9 proteins co-purify in the absence of crRNA and tracrRNA.

(E) SDS-PAGE of the purified complex used for structural analysis.