In contrast, at the 5-s timepoint of the NTS mapping experiments for WT AsCas12a, the two distributions were almost completely non-overlapping, having already developed peaks at the major cut sites (Appendix 2—figure 1B, Appendix 2—figure 2—figure supplement 10). 4 µL 160 mM KMnO4 (solution prepared in permanganate reaction buffer immediately before reaction) was added and allowed to react for 2 min (unless otherwise indicated) at 30°C. Envelope surface area (ESA) was determined by isolating two nucleobases of the interhelical stack—that on the RNA terminus and that stacked upon it from the NTS—and calculating the surface area of the volume they jointly occupy over the course of each trajectory (envelope shown in cyan). We agree that protein binding energy is probably mechanistically important, and we agree that our data have not definitively established a causative link between DNA junction stability and target-strand cleavage. According to these mapping experiments, the non-target strand has two major cleavage sites, at dinucleotides 13/14 and 18/19 (numbers denote distance from the PAM), suggesting the formation of a 5-nt gap within the tract of DNA displaced by the crRNA (Appendix 2—figure 1B). We also observed this effect for a second set of crRNA/DNA sequences with equivalent base-pairing topology, demonstrating that this result is not unique to the originally tested sequence (Figure 3—figure supplement 2). Section about "difference in interhelical stacking energy may underlie asymmetric R-loop flank stability": Here the authors "hypothesize that the asymmetry may emerge from energetic differences in the coaxial stacking of a DNA homoduplex on either end of an RNA:DNA hybrid", which got me lost. In other words, out of all the DNA molecules on the gel for which oxidation of thymine 1 would have been observable (i.e., cleavage fragments at or above the thymine-1 fragment on the gel), what fraction of those molecules were in fact oxidized at thymine 1? To test the hypothesis that distortion in the R-loop flank is linked to RuvC-mediated target-strand cleavage, we assembled wild type (WT) Cas12a with R-loops of various sizes and determined the distribution of target-strand cut sites. The difference in the stacking equilibrium then leads to an unequal propensity for base pairing in the flanking DNA homoduplex. TS cleavage was almost undetectable in the presence of a single nick, and its extent of cleavage only reached that observed with a fully phosphodiester-linked NTS when the gap was widened to 5 nt (Appendix 2—figure 2A, Appendix 2—figure 2—figure supplement 5). Phosphorimage of AsCas12a cleavage products, resolved by denaturing PAGE.
To precisely determine the location of Cas12a-catalyzed NTS cleavage, we monitored the formation of DNA cleavage products over time by denaturing PAGE, and we distinguished between different DNA fragments by placing radiolabels on the 5′ or 3′ end of each strand (Appendix 2—figure 1, Appendix 2—figure 1—figure supplement 1, Figure 2—figure supplement 1). Beyond uncertainty in the measurement, it is also unknown to what extent the probing technique is changing the fundamental biophysical features of the DNA structures. When we probed the permanganate reactivity of this protein-free R-loop, we found that the A/T tract was slightly more reactive than in the Cas12a-generated R-loop experiment (Figure 4A, Figure 4—figure supplement 1). Kevin Pho, MD shares the stories of the many who intersect with our health care system but are rarely heard from. Boster's got them all.
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