(A) Using an EMSA, we examined the gel shift experienced by a 36 bp target dsDNA, fluorescently labeled with a 6-FAM marker and incubated with either purified SpyCas9, sgRNA, and/or purified Acrs. Divalent cations were omitted from reactions such that SpyCas9 could bind, but not cleave, target DNA, as previously shown (Lee et al., 2018). SpyCas9 did not bind the shorter dsDNA target used in these experiments as proficiently as it did the 60 bp oligo used in Figure 6A, explaining the fainter SpyCas9-bound bands. Lane numbers and reaction components are depicted below the native gel. Acr concentrations are depicted above the gel in brackets, relative to SpyCas9 (which was used at 2.25 µM). Key bands are annotated to the left of the gel. (B) A Western blot of the same gel depicted in (A), probing for SpyCas9. Gel and blot images were manually aligned using well and lane delineators, cropped identically, and depicted next to one another. Thus, the SpyCas9 migration in (B) is directly comparable to the dsDNA migration depicted in (A). No SpyCas9 co-migrates with band #2, seen in (A). Taken together, (A) and (B) indicate that AcrIIA11 binds the SpyCa9/sgRNA/dsDNA ternary complex, binds dsDNA but not ssDNA, and is stimulated to bind dsDNA in the presence of SpyCas9. (C) SpyCas9 was incubated with Acrs at varying concentrations with and without sgRNA, run through a native gel, and a Western Blot against SpyCas9 was performed. Independent of sgRNA, AcrIIA11 restricts the migration of SpyCas9 through the native gel in a concentration dependent manner, suggestive of binding. Lane numbers and reaction components are depicted below the native gel. Acr concentrations are depicted above the gel in brackets, relative to SpyCas9 (which was used at 2 µM). The gel used in (C) was run for a longer time than the gel used in (B), explaining the extended SpyCas9 migration in (C). (D) A native gel was run exactly as in (C) but stained for total protein content with Coomassie dye rather than used for a Western blot. Much of the Coomassie-stained protein in (D) matches the SpyCas9 signal in (C), with additional bands that correspond to AcrIIA11 (indicated at right). In the presence of SpyCas9, the AcrIIA11 band appears brighter, perhaps indicating stabilization of a conformation or oligomer state of the protein. Protein is visible in an AcrIIA11-only control (lane 10) in (D) at the same position at which SpyCas9’s migration is restricted in (C) (lanes 4 and 12).