Fig. 3: Observation and quantification of terminal bead loss for targeted DNA regions with canonical PAM spacing in the absence or presence of RNAP.
From: Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation
a, Typical DNA extension time-trace, obtained using S2_T0 SpCas9 combined with RNAP. DNA was initially positively supercoiled by five turns to allow injection of the reagent to the sample chamber under conditions in which the reaction is inhibited, after which the DNA was unwound by eight negative turns (clockwise arrow) to permit the sequence of events including R-loop formation, supercoil loss (blue arrow), and terminal bead loss (as per Extended Data Fig. 7b). The moment of terminal bead loss is represented above the time-trace as a magnetic bead topped by an up arrow. The time-trace y-axis is labelled such that the maximal bead position above the surface is set to zero, and lower extensions are reported as negative values. b, The percentage of bead-loss events, normalized by the number of molecules that formed R-loops (as per Extended Data Fig. 7b), shows a bimodal distribution. c,d, As in a and b, but for S3_T0 SpCas9 without RNAP. No bead loss was observed on this time-trace. e,f, As in c and d, but for S3_T0 SpCas9 with RNAP. The mean bead-loss fractions observed within a 2-h window for S2_T0 SpCas9 plus RNAPs, S3_T0 SpCas9, and S3_T0 SpCas9 plus RNAP are 56.5% (n = 69), 22% (n = 59), and 52.4% (n = 103), respectively. All events lasting for more than 10,800 s are reported as 10,800 s. See Supplementary Table 3 for detailed values.
