Figure 2. FACT Complex Interacts with DNA-Bound Cas9 and dCas9 to Promote Eviction and Multi-Turnover Behavior.

(A) Schematic of samples prepared for mass spectrometry. dCas9-BirA* programmed with the on-target gRNA, Cas9-BirA* programmed with the on-target guide, and dCas9-BirA* programmed with a NT sgRNA were incubated with a 10-fold molar excess of plasmid and then added to HSS containing the ARS and biotin. Biotinylated proteins were isolated with Streptavidin-coupled beads and identified through mass spectrometry.

(B) Volcano plot of biotinylated proteins in Cas9-BirA*–on-target gRNA samples (N = 3 biological replicates) versus dCas9-BirA*–NT gRNA samples (N = 3 biological replicates). Colored circles correspond to factors that were significantly enriched (p < 0.05) according to a Limma analysis. Red circles correspond to the two components of the FACT complex. See also Figure S1D and Table S1.

(C) Enrichment of biotinylated SSRP1 and SPT16 in HSS containing dCas9-BirA*–on-target gRNA versus dCas9-BirA*–NT gRNA.

(D) FACT immunodepletion inhibits dCas9 eviction in HSS. A 10-fold molar excess of Cas9 was added to dCas9 RNP-plasmid complexes incubated in SSRP1 or SPT16-immunodepleted extract either in the presence or absence of recombinant FACT. See also Figure S1E.

(E) FACT promotes Cas9’s multi-turnover activity. Cas9 RNPs were incubated with a plasmid substrate in a 1:2 molar ratio with either buffer, mock-immunodepleted HSS with ATP, SSRP1-immunodepleted HSS with ATP, or HSS with CIP and ATPγS for 180 min.

(F) Recombinant FACT displaces dCas9 pre-loaded on a plasmid in a minimal buffer system.