The study presents the development of an evolved Cas9 variant, xCas9, which has broadened PAM compatibility and improved DNA specificity. Using phage-assisted continuous evolution (PACE), the researchers created xCas9 variants that can recognize a wider range of PAM sequences, including NG, GAA, and GAT, in addition to the commonly used NGG PAM. This expansion of PAM compatibility is the broadest reported for Cas9s active in mammalian cells. xCas9 variants were characterized for their activity in human cells, demonstrating enhanced transcriptional activation, DNA cleavage, and base editing capabilities compared to the wild-type SpCas9. Notably, xCas9 variants showed significantly reduced off-target activity, even with their broader PAM compatibility. These findings expand the DNA targeting scope of CRISPR systems and demonstrate that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity.The study presents the development of an evolved Cas9 variant, xCas9, which has broadened PAM compatibility and improved DNA specificity. Using phage-assisted continuous evolution (PACE), the researchers created xCas9 variants that can recognize a wider range of PAM sequences, including NG, GAA, and GAT, in addition to the commonly used NGG PAM. This expansion of PAM compatibility is the broadest reported for Cas9s active in mammalian cells. xCas9 variants were characterized for their activity in human cells, demonstrating enhanced transcriptional activation, DNA cleavage, and base editing capabilities compared to the wild-type SpCas9. Notably, xCas9 variants showed significantly reduced off-target activity, even with their broader PAM compatibility. These findings expand the DNA targeting scope of CRISPR systems and demonstrate that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity.