Mechanism of expanded DNA recognition in xCas9

xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. Thi..., The dataset was created using molecular dynamics simulations of SpCas9 and xCas9 proteins. Data has been processed using standard Python libraries and the GROMACS. , , # xCas9: Molecular dynamics [https://doi.org/10.5061/dryad.0000000dt](https://doi.org/10.5061/dryad.0000000dt) ## Description of the data and file structure Processed source data are available in text and XLSX formats, corresponding to each Figure (Fig. 2 to 5 and their supplementary Figs in the article). The corresponding MD trajectories (each replica) are deposited in XTC format, with structures in PDB format. They can be visualized together using programs such as VMD and PyMOL. The TPR files can be used to conduct or verify analyses using GROMACS. **Description of each file or subdirectory:** 1. `Figure_2-source_data_1_for_panelB`: Data represent interaction pattern established by R1333 and R1335 with PAM nucleobases (NB), PAM backbone (BB) and non-PAM nucleotides in SpCas9 bound to TGG (i.e., the wilt-type system) and xCas9 bound to PAM sequences that are recognized (TGG, AAG, GAT) and ignored (CCT, TTA, ATC). 2. `Figure_2-source_data_2_for_panelD`: Data of frequencies of hyd...