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. Author manuscript; available in PMC: 2016 Oct 15.
Published in final edited form as: Mol Cell. 2015 Oct 15;60(2):242–255. doi: 10.1016/j.molcel.2015.09.020

Figure 2. Strand Specificity of NmeCas9’s Two Nuclease Domains.

Figure 2

(A) Schematic representation of the location of the RuvC and HNH domains in the primary sequence of NmeCas9.

(B) Plasmid cleavage assays were performed using wildtype NmeCas9, as well as active site mutants D16A, H588A, and D16A+H588A. N, nicked; L, linearized; SC: supercoiled.

(C) NmeCas9 uses the HNH and RuvC nuclease domains to cleave the crRNA-complementary and non-complementary strands, respectively. The oligonucleotide cleavage assay was performed using tracrRNA- and crRNA-programmed, wildtype and mutant NmeCas9 proteins at 37°C for 30 min. Duplex DNA substrates were 5′ 32P labeled on either strand. M, size markers. The sizes of the substrates, cleavage products and markers are indicated. The single and double asterisks denote non-complementary strand and complementary strand (respectively) minor cleavage products that are abolished when either active site is mutated. The significance (if any) of these minor cleavage products is not clear.