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. Author manuscript; available in PMC: 2018 Mar 29.
Published in final edited form as: Cell. 2017 Aug 24;170(6):1224–1233.e15. doi: 10.1016/j.cell.2017.07.037

Figure 3. AcrIIC1 binds to Cas9 HNH domain.

Figure 3

(A) Domain schematics of GeoCas9 truncations and Cas9 chimeras, designed to identify the Cas9 binding interface of AcrIIC1. Constructs 1-10 were incubated with AcrIIC1, fractionated over an S200 size-exclusion column and analyzed by SDS-PAGE. Constructs that bound to AcrIIC1 are indicated with a (+) and constructs that showed no interaction are indicated with a (−). The chimeric Cas9 proteins (7-10) were generated by switching the HNH domains of a Cas9 that is not inhibited by AcrIIC1 (AnaCas9) and a Cas9 that is inhibited (GeoCas9).

(B) Fractions from the S200 runs in Figure S3A were separated on a 4-20% SDS-PAGE gel. Numbers above the gel correspond to the construct or chimera numbers from Figure 2A.

(C) Elution from an S75 size exclusion column of NmeCas9 HNH domain (purple), AcrIIC1 (orange) or the two incubated together with 2-fold excess AcrIIC1(red). See also Figure S3.