Fig. 3.

PAM attachment efficiencies. (A) Sequence of the PAM site occupancy library. (B) Measured PAM occupancies for all six-base PAM sites normalized by the PAM occupancy of the (T)TTTV consensus sequence. V = C, G, or A. Error bars = aggregated LOESS fit of the mean/variance relationship between experimental replicates of the fold change. (C) Aggregated PAM site occupancies display a preference for the (T)TTTV consensus sequence while still allowing binding to occur for TTV sequences. Error bars = SD. (D) Bit content and (E) probability density of the SK-selected PAM site libraries show a strong preference for NNNTTV PAM sites. N = T, C, G, or A. (F) Predicted $\theta \left(PAM\right)$ using the base-dependent binding energy expression ${ϵ}_{PAM}={\sum }_i{ϵ}_b^i$ shows a strong agreement with the measured occupancies (Pearson correlation = 0.943). (G) Fitted values for position- and base-dependent binding energies in units of $k_{B}T$ for PAM sites of the form TTV ($k_B$ = Boltzmann constant, T = temperature). Details about the model and an expression for the binding energies for (T)TTTV PAM sites are included in SI Appendix. Note that this model only considers PAM sequences of the form NNNTTN by constraining ${ϵ}_V^{-2},{ϵ}_V^{-3}\to \infty$.