Figure 5.

RNA-cleavage activity of the TtCmr complex. (A) An internally labeled RNA substrate complementary to CRISPR-4.5 was incubated with the endogenous Cmr complex for 1 h in the presence of different concentrations of Mg²⁺. Samples were analyzed by denaturing PAGE, followed by phosphorimaging. (B) Different internally labeled RNA targets (complementary to CRISPR-4.5, 4.2, 2.1 and 3.2) were tested in a similar assay (including 2 mM Mg²⁺), demonstrating the specificity of the Cmr complex. (C) Activity assay with the endogenous Cmr complex using a 5’ or 3’ labeled RNA substrate (CRISPR-4.5). (D) The 5’ labeled RNA substrate (CRISPR-4.5) was incubated with either the endogenous (“Cmr”) or the reconstituted Cmr complex (“Rec. Cmr”) for the indicated amount of time. The reconstituted complex was pre-loaded with the 43 or the 46 nt crRNA. Reconstituted Cmr complexes without crRNA (“-“) were included as a control. Noncontiguous lanes from the same gel are indicated with dotted lines. The results of a Cmr activity assay with complementary ssDNA and dsDNA substrates are depicted in Fig. S5.