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. Author manuscript; available in PMC: 2016 Nov 29.
Published in final edited form as: Science. 2016 Jun 2;353(6299):aaf5573. doi: 10.1126/science.aaf5573

Figure 2. LshC2c2 and crRNA mediate RNA-guided ssRNA cleavage.

Figure 2

A) Schematic of the ssRNA substrate being targeted by the crRNA. The protospacer region is highlighted in blue and the PFS is indicated by the magenta bar.

B) A denaturing gel demonstrating crRNA-mediated ssRNA cleavage by LshC2c2 after 1 hour of incubation. The ssRNA target is either 5’ labeled with IRDye 800 or 3’ labeled with Cy5. Cleavage requires the presence of the crRNA and is abolished by addition of EDTA. Four cleavage sites are observed. Reported band lengths are matched from RNA sequencing.

C) A denaturing gel demonstrating the requirement for an H PFS (not G) after 3 hours of incubation. Four ssRNA substrates that are identical except for the PFS (indicated by the magenta X in the schematic) were used for the in vitro cleavage reactions. ssRNA cleavage activity is dependent on the nucleotide immediately 3’ of the target site. Reported band lengths are matched from RNA sequencing.

D) Schematic showing five protospacers for each PFS on the ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity after 1 hour of incubation. crRNAs correspond to protospacer numbering. Reported band lengths are matched from RNA sequencing.