A key limitation of CRISPR-Cas9 for genome editing is the requirement of a specific protospacer adjacent motif (PAM), primarily NGG for Streptococcus pyogenes (SpCas9). This restricts targeting in mammalian cells. Researchers used phage-assisted continuous evolution (PACE) to evolve an expanded PAM SpCas9 variant (xCas9) that recognizes a broader range of PAM sequences, including NG, GAA, and GAT. xCas9 exhibits broader PAM compatibility and higher DNA specificity than SpCas9, with significantly reduced off-target activity. This expansion of PAM compatibility and DNA specificity enhances the utility of CRISPR systems in mammalian cells for applications such as transcriptional activation, gene disruption, and base editing. xCas9 variants show improved efficiency in targeting diverse PAM sequences, including non-NGG PAMs, and demonstrate enhanced DNA specificity compared to SpCas9, with reduced off-target activity. These findings indicate that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity. xCas9 variants also show improved performance in base editing, including cytidine and adenine base editing, and are compatible with various CRISPR-based applications. The results highlight the potential of xCas9 to expand the scope of genome editing in mammalian cells and support the development of more efficient and specific genome editing tools.A key limitation of CRISPR-Cas9 for genome editing is the requirement of a specific protospacer adjacent motif (PAM), primarily NGG for Streptococcus pyogenes (SpCas9). This restricts targeting in mammalian cells. Researchers used phage-assisted continuous evolution (PACE) to evolve an expanded PAM SpCas9 variant (xCas9) that recognizes a broader range of PAM sequences, including NG, GAA, and GAT. xCas9 exhibits broader PAM compatibility and higher DNA specificity than SpCas9, with significantly reduced off-target activity. This expansion of PAM compatibility and DNA specificity enhances the utility of CRISPR systems in mammalian cells for applications such as transcriptional activation, gene disruption, and base editing. xCas9 variants show improved efficiency in targeting diverse PAM sequences, including non-NGG PAMs, and demonstrate enhanced DNA specificity compared to SpCas9, with reduced off-target activity. These findings indicate that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity. xCas9 variants also show improved performance in base editing, including cytidine and adenine base editing, and are compatible with various CRISPR-based applications. The results highlight the potential of xCas9 to expand the scope of genome editing in mammalian cells and support the development of more efficient and specific genome editing tools.