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A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

Fig 4

Parameterized free energy models show how mismatched crRNA guide sequences and DNA site sequences affect Cas9 cleavage activity.

The (A) 21 position-dependent and (B) 256 sequence-dependent free energy model coefficients were determined using either (left) 3671 in vitro Cas9 cleavage measurements from dataset I or the (right) 5979 in vivo Cas9 cleavage measurements from dataset II. Coefficients were normalized to their maximum values. White boxes show unidentifiable model parameters, based on the available measurements. (C) Comparisons between apparent and model-calculated ΔΔGexchange across all single measurements. Pearson R2 is 0.74 and 0.61, respectively. All points represent single measurements from Pattanayak et. al., Hsu et. al., and Mali et. al [33,37,41]. (D) An example showing how the model is used to calculate ΔΔGexchange and ΔGPAM for a specific guide RNA sequence and DNA site. The energetic contributions of the three mismatches are determined by their (A) position-dependent coefficients and their (B) dinucleotide RNA:DNA identities, using the model parameterized by (left) dataset I. The (green box) PAM sequence determines ΔGPAM using Table 3.

Fig 4