Fig. 1: High-resolution maps of dCas interactions with DNA using the DNA unzipping mapper.
a, DNA unzipping mapper configuration. An unzipping template is tethered at one end to the surface of a coverslip of a sample chamber and at the other end to a polystyrene bead held in an optical trap. Using the optical trap, the bead is moved relative to the surface, progressively unzipping the DNA until the unzipping fork reaches a bound protein, which resists unzipping, leading to a distinct rise in force. The location of the rise in force is used to map the protein location. b, Representative unzipping traces (red) of bound dCas9 (top), bound dCas12a (middle) and a paused TEC (bottom), along with naked DNA traces (black). The DNA was unzipped from either direction (black arrows) relative to the bound protein for each protein. Two conformations were detected when a bound dCas12a protein was unzipped from the PAM-distal side, shown as light blue and red. The two dashed lines bracket the expected gRNA-DNA hybrid locations for dCas9 or dCas12a and the expected RNA-DNA hybrid of a TEC. The red arrows indicate locations where the unzipping force dipped below the naked DNA baseline. c, Hypothesized mechanism for transcription read-through from the PAM-distal side. Note that gRNA hybridizes with the TEC template and nontemplate strand for a bound dCas9 and dCas12a complex, respectively. Source data containing traces for b are provided.
