Figure 1.
Quantitative Analysis of Cas9-Induced DSBs
(A) Proposed model of DSB repair based on stochastic transitions between intact, broken, and indel states. kp and km are rate constants of perfect and mutagenic repair, respectively; kc is the rate constant of cutting by Cas9. The latter depends on Cas9 activation and is therefore denoted as kc(t).
(B) ODEs describing the three reaction steps, with rate constants as parameters.
(C) Outline of the experimental strategy; see main text.
(D) Representative time course experiment, showing gradual accumulation of indels. A sigmoid curve was fitted to the data to determine the plateau level at late time points (dashed line), which reflects the transfection efficiency.
(E) Relative proportions of intact (red) and indel (green) fractions at the LBR2 locus over time. The data points are normalized on to the total indel fraction to correct for the variation in transfection efficiency. Indel fraction in absence of Shield-1 is shown in gray. Average of 7 independent experiments is shown; error bars represent the SD, and the dashed lines show fitted sigmoid curves.
(F) Western blot analysis of Cas9 presence as a function of time. Tubulin was used as loading control.
(G) The intensities of Cas9 antibody signal were determined by densitometry from time points of 3 individual western blots and normalized to a sample incubated for 60 hr with Shield-1 (lanes labeled “K562#17+Shield-1” in F). An ODE fit was performed to determine the activity score of Cas9 in time.