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. Author manuscript; available in PMC: 2018 Jul 6.
Published in final edited form as: Mol Cell. 2017 Jun 9;67(1):117–127.e5. doi: 10.1016/j.molcel.2017.05.024

Figure 1. AcrIIA4 directly interacts with sgRNA-bound SpyCas9 and inactivates SpyCas9.

Figure 1

(A) In vitro enzymatic assay monitoring cleavage of linear dsDNA by SpyCas9 and sgRNA in the presence of AcrIIA4. The molar ratios of AcrIIA4:SpyCas9 are shown at the top of each lanes (left panel). The inhibition between MBP-tagged SpyCas9 and AcrIIA4 are also detected and compared with dSpyCas9 (right panel).

(B) AcrIIA4 selectively forms a stable complex with sgRNA-bound SpyCas9 rather than apo or DNA-bound SpyCas9-sgRNA in solution. SEC was performed using SpyCas9 in the presence or absence of sgRNA and sgRNA-dsDNA.

(C) AcrIIA4 physically interacts with sgRNA-bound SpyCas9. MBP pull-down assays were performed using MBP-tagged AcrIIA4 and SpyCas9 in presence or absence of sgRNA and sgRNA-dsDNA.

(D) Oligomeric state of AcrIIA4 in solution detected by SEC-MALS. The horizontal red line represents the SEC-MALS calculated mass for AcrIIA4. The calculated and theoretical molecular masses are 10.4 kDa and 10.2 kDa, respectively, indicating that AcrIIA4 exists as a monomer in solution.

See also Figure S1.