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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 2. AcrIIC1 traps the DNA-bound Cas9 complex.

Figure 2

(A) Cartoon of Cas9-mediated double-stranded DNA cleavage. Guide RNA (black) is duplexed to the DNA target strand (red), which is splayed from the DNA non-target strand (blue) adjacent to the PAM sequence (yellow). The HNH and RuvC nuclease domains (black triangles) cleave the target strand and non-target strand, respectively.

(B) Radiolabeled cleavage assays conducted using GeoCas9 to measure AcrIIC1 inhibition of cleavage on the target and non-target strands. Cas9–sgRNA RNP was complexed with or without AcrIIC1 and added to radiolabeled target DNA duplex with each strand labeled separately. The lanes for a given condition correspond to increasing time (0-30min) from left to right. Black triangles indicate cleavage products.

(C) Analysis of GeoCas9 binding and cleavage in the presence or absence of AcrIIC1 analyzed on a non-denaturing gel with the non-target strand labeled. GeoCas9 RNP concentration was varied in the absence or presence of excess AcrIIC1. The top band corresponds to GeoCas9 bound to the target DNA, the middle band is free DNA, and the lower band is cleaved DNA (concentration series correspond to 0, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512nM of GeoCas9 RNP). See also Figure S2.