The study addresses the limitation of CRISPR-Cas9 nucleases, which are widely used for genome editing, due to their restricted ability to recognize specific protospacer adjacent motifs (PAMs). The researchers engineered *Streptococcus pyogenes* Cas9 (SpCas9) to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. They identified and characterized variants of SpCas9 that can robustly edit endogenous gene sites in zebrafish and human cells, which are not targetable by wild-type SpCas9. These variants have comparable genome-wide specificities to wild-type SpCas9, as determined by GUIDE-Seq analysis. Additionally, they identified a SpCas9 variant with improved specificity in human cells, which better discriminates against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. The researchers also found that two smaller-size Cas9 orthologues, *Streptococcus thermophilus* Cas9 (StCas9) and *Staphylococcus aureus* Cas9 (SaCas9), function efficiently in bacterial selection systems and human cells, suggesting that their PAM specificities can be modified. These findings provide useful SpCas9 variants and establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.The study addresses the limitation of CRISPR-Cas9 nucleases, which are widely used for genome editing, due to their restricted ability to recognize specific protospacer adjacent motifs (PAMs). The researchers engineered *Streptococcus pyogenes* Cas9 (SpCas9) to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. They identified and characterized variants of SpCas9 that can robustly edit endogenous gene sites in zebrafish and human cells, which are not targetable by wild-type SpCas9. These variants have comparable genome-wide specificities to wild-type SpCas9, as determined by GUIDE-Seq analysis. Additionally, they identified a SpCas9 variant with improved specificity in human cells, which better discriminates against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. The researchers also found that two smaller-size Cas9 orthologues, *Streptococcus thermophilus* Cas9 (StCas9) and *Staphylococcus aureus* Cas9 (SaCas9), function efficiently in bacterial selection systems and human cells, suggesting that their PAM specificities can be modified. These findings provide useful SpCas9 variants and establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.