Extended Data Fig. 9: Apparent efficiency of DSB formation via magnetic trapping microscopy as determined by terminal loss of the magnetic bead within a three-hour window.
From: Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation
(a) Raw percentages of all observed DNA molecules displaying terminal bead loss in assays including S1_T0 SpCas9 (45.2 ± 4.9% SEM or 47 of n = 104 molecules), S1_T1 SpCas9 (89.5 ± 3.1% SEM, or 85 of n = 95 molecules), S1_T2 SpCas9 (77.2 ± 4.7% SEM, or 61 of n = 79 molecules), S2_T0 SpCas9 (27.3 ± 5.1% SEM, 21 of n = 77 molecules) and S3_T0 SpCas9 (23.1 ± 5.2% SEM, or 15 of n = 65 molecules) (blue bars). Experiments combining S2-T0 SpCas9 and RNAP (58.7 ± 5.7% SEM, or 44 of n = 75 molecules), and S3_T0 SpCas9 and RNAP (52.3 ± 4.8% SEM, or 56 of n = 107 molecules) are also quantified (orange bars). (b) Normalized percentages of those R-loop-forming DNA molecules which go on to display terminal bead loss for: S1_T0 SpCas9 (55.1 ± 5.3%, n = 89), S1_T1 SpCas9 (97.7 ± 1.6%, n = 86), S1_T2 SpCas9 (93.8 ± 3.0%, n = 64), S2_T0 SpCas9 (27.6 ± 5.1%, n = 76) and S3_T0 SpCas9 (23.7 ± 5.5%, n = 59), are normalized by dividing the number of molecules that formed R-loops (cyan bars with SEMs). Similarly, the normalized percentages for S2_T0 SpCas9 plus RNAP (64.3 ± 5.7%, n = 70) and NT00-SpCas9 plus RNAP (59.4 ± 5.0%, n = 96) are shown in yellow bars. SEM was derived from the binomial distribution with a 95% confidence interval.
