A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation
(A) Model-calculated target binding free energies (ΔGtarget) are shown across genome position, plotting only one in ten positions for improved visualization. Panels represent either the (top, blue) forward strand or (bottom, red) reverse strand of the λ-phage genome. The target binding free energies are the sum of (B) the free energy change when dCas9 binds to a PAM site (ΔGPAM), (C) the free energy change during R-loop formation at PAM-proximal sites, compared to a perfectly complementary sequence (ΔΔGexchange), and the free energy change as a result of varying DNA site superhelical density (ΔΔGsupercoiling). The major on-target site λ2 is denoted by stars. A major off-target site OS1 is denoted by crosses. Here, each mismatch in the crRNA and DNA site sequences contributes up to 0.78 kcal/mol to ΔΔGexchange, depending on their distance from the PAM site. The λ-phage genome is assumed to have uniform DNA superhelical density. The model-calculated binding probabilities of (d)Cas9:crRNAλ2 to all possible PAM sites are shown at (D) the initial time before any Cas9 activity or (F) after a 10 minute incubation with (d)Cas9:crRNAλ2. (E) We show the model-calculated dynamics of (d)Cas9 binding occupancy at the (black line) λ2 DNA site, the (green line) major off-target site OS1, and a (inset) single off-target site with ΔGtarget = 0 kcal/mol.