Fig 4. RNA-guided ssDNA cleavage by TthCsm.

(A) Schematic of the DNA cleavage reaction. TthCsm was mixed with 5′-³²P-labeled ssDNA oligonucleotide and an unlabeled complementary ssRNA oligonucleotide in the presence of 1 mM EDTA. The reaction was initiated by the addition of 5 mM MnCl₂. (B) TthCsm-mediated ssDNA cleavage was tested in the presence of complementary (C) or noncomplementary (NC) ssRNA, as described in (A). Time points were taken at 0, 10, 15, and 30 min, and cleavage products were analyzed by denaturing PAGE. (C) As in (B), but using unlabeled ssRNA substrates that are complementary to the crRNA guide sequence, but have an 8 nt-long 3′ flanking region that is either noncomplementary (3′-nc), complementary (3′-c), truncated (Δ3′), or both extended to 20 nt long and noncomplementary (3′-ext).