This study describes the engineering of CRISPR-Cas9 nucleases with altered protospacer adjacent motif (PAM) specificities. The commonly used Streptococcus pyogenes Cas9 (SpCas9) was modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enabled robust editing of endogenous gene sites in zebrafish and human cells not targetable by wild-type SpCas9, with genome-wide specificities comparable to wild-type SpCas9. Additionally, a SpCas9 variant with improved specificity in human cells was identified, showing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. Two smaller Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), were found to function efficiently in bacterial selection systems and human cells, suggesting that the engineering strategies could be extended to Cas9s from other species. The findings provide useful SpCas9 variants and establish the feasibility of engineering Cas9s with altered and improved PAM specificities. The study also demonstrates that the VQR and VRER variants enable modification of previously inaccessible sites in zebrafish embryos and human cells, and computational analysis of the reference human genome reveals that they double the targeting potential of SpCas9. The study also identifies target sites for the engineered variants using a web-based tool called CasBLASTR. The results show that the VQR and VRER variants have similar or better genome-wide specificities compared to wild-type SpCas9. These variants can be rapidly incorporated into existing SpCas9 vectors by simple site-directed mutagenesis. The study also demonstrates that the D1135E substitution increases the specificity of SpCas9. The study also shows that St1Cas9 and SaCas9 function in human cells, making them attractive candidates for engineering additional variants with novel PAM specificities. The study provides a comprehensive understanding of the structural and functional roles of D1135, G1218, and T1337 in PAM recognition by SpCas9. The study also describes the bacterial-based positive selection assay for evolving SpCas9 variants and the bacterial-based site-depletion assay for profiling Cas9 PAM specificities. The study also describes the human cell culture and transfection methods, zebrafish care and injections, human cell EGFP disruption assay, T7E1 assay, targeted deep-sequencing, and GUIDE-seq to quantify nuclease-induced mutations. The study also includes extended data figures and tables providing additional information on the study.This study describes the engineering of CRISPR-Cas9 nucleases with altered protospacer adjacent motif (PAM) specificities. The commonly used Streptococcus pyogenes Cas9 (SpCas9) was modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enabled robust editing of endogenous gene sites in zebrafish and human cells not targetable by wild-type SpCas9, with genome-wide specificities comparable to wild-type SpCas9. Additionally, a SpCas9 variant with improved specificity in human cells was identified, showing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. Two smaller Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), were found to function efficiently in bacterial selection systems and human cells, suggesting that the engineering strategies could be extended to Cas9s from other species. The findings provide useful SpCas9 variants and establish the feasibility of engineering Cas9s with altered and improved PAM specificities. The study also demonstrates that the VQR and VRER variants enable modification of previously inaccessible sites in zebrafish embryos and human cells, and computational analysis of the reference human genome reveals that they double the targeting potential of SpCas9. The study also identifies target sites for the engineered variants using a web-based tool called CasBLASTR. The results show that the VQR and VRER variants have similar or better genome-wide specificities compared to wild-type SpCas9. These variants can be rapidly incorporated into existing SpCas9 vectors by simple site-directed mutagenesis. The study also demonstrates that the D1135E substitution increases the specificity of SpCas9. The study also shows that St1Cas9 and SaCas9 function in human cells, making them attractive candidates for engineering additional variants with novel PAM specificities. The study provides a comprehensive understanding of the structural and functional roles of D1135, G1218, and T1337 in PAM recognition by SpCas9. The study also describes the bacterial-based positive selection assay for evolving SpCas9 variants and the bacterial-based site-depletion assay for profiling Cas9 PAM specificities. The study also describes the human cell culture and transfection methods, zebrafish care and injections, human cell EGFP disruption assay, T7E1 assay, targeted deep-sequencing, and GUIDE-seq to quantify nuclease-induced mutations. The study also includes extended data figures and tables providing additional information on the study.