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. Author manuscript; available in PMC: 2017 Nov 29.
Published in final edited form as: ACS Synth Biol. 2017 Mar 20;6(6):1103–1113. doi: 10.1021/acssynbio.7b00050

Figure 2.

Figure 2

Experimental confirmation of the 5′-NNNCC-3′ PAM for AceCas9 on oligo DNA substrates. (A) Cleavage results of double stranded DNA oligos (dsDNA) containing the 5′-NNNCC-3′ PAM. (Left) examination of HEX-labeled targeting DNA strand and its annealed dsDNA product in a nondenaturing gel. (Right) cleavage of dsDNA labeled with fluorophore 6-FAM on its nontargeting strand (red) and cleavage of dsDNA labeled with fluorophore HEX on its targeting strand (green). (B) Cleavage results of dsDNA oligos containing variations in PAM. (Top) Sequences and names of the wild-type and mutant oligo DNA. Mutations are indicated in red. (Bottom) Results of oligo DNA cleavage visualized on a 15% denaturing polyacrylamide gel. The two wild-type (WT) dsDNA are distinguished by the position of the fluorescence labels. The dsDNA with nontargeting strand labeled by 6-FAM is denoted as WT*, whereas that with targeting strand labeled by HEX is denoted as WT. Other dsDNA variants are indicated by nucleotides in red.