Cas9, derived from CRISPR-Cas systems, has revolutionized genome editing by enabling precise and efficient modifications in diverse organisms. This review highlights the versatility of Cas9 as a programmable tool for engineering biology, capable of targeting, regulating, and modifying genomes. Cas9, an RNA-guided DNA-binding protein, can simultaneously localize RNA, DNA, and protein, offering unprecedented control over cellular processes. The CRISPR-Cas system in bacteria and archaea uses short RNA to direct the degradation of foreign nucleic acids. Type II CRISPR-Cas systems have been engineered to enable robust RNA-guided genome modifications in eukaryotic systems, significantly improving genome editing and regulation. Cas9 can be modified to function as a nuclease-null protein, allowing for the targeting of specific DNA sequences without cleavage, enabling the fusion of proteins and RNAs to any DNA sequence. This capability allows for the precise regulation of gene expression, including activation and repression of genes, and the modulation of epigenetic marks and genome architecture. Cas9 can also be used to engineer recombinases and transposases, enabling targeted genome modifications. The use of sgRNA libraries allows for the simultaneous targeting of multiple genes, facilitating the study of complex biological processes. However, challenges remain in improving the specificity and efficiency of Cas9-based genome editing, particularly in reducing off-target effects. The development of more specific Cas9 variants and the optimization of sgRNA design are critical for advancing the therapeutic and research applications of CRISPR-Cas technology. The potential of Cas9 in therapeutic applications, such as correcting genetic disorders and modifying gene expression, is significant, although challenges in delivery and specificity must be addressed. Overall, Cas9 represents a powerful tool for genome engineering, with broad implications for basic science, cellular engineering, and therapeutic applications.Cas9, derived from CRISPR-Cas systems, has revolutionized genome editing by enabling precise and efficient modifications in diverse organisms. This review highlights the versatility of Cas9 as a programmable tool for engineering biology, capable of targeting, regulating, and modifying genomes. Cas9, an RNA-guided DNA-binding protein, can simultaneously localize RNA, DNA, and protein, offering unprecedented control over cellular processes. The CRISPR-Cas system in bacteria and archaea uses short RNA to direct the degradation of foreign nucleic acids. Type II CRISPR-Cas systems have been engineered to enable robust RNA-guided genome modifications in eukaryotic systems, significantly improving genome editing and regulation. Cas9 can be modified to function as a nuclease-null protein, allowing for the targeting of specific DNA sequences without cleavage, enabling the fusion of proteins and RNAs to any DNA sequence. This capability allows for the precise regulation of gene expression, including activation and repression of genes, and the modulation of epigenetic marks and genome architecture. Cas9 can also be used to engineer recombinases and transposases, enabling targeted genome modifications. The use of sgRNA libraries allows for the simultaneous targeting of multiple genes, facilitating the study of complex biological processes. However, challenges remain in improving the specificity and efficiency of Cas9-based genome editing, particularly in reducing off-target effects. The development of more specific Cas9 variants and the optimization of sgRNA design are critical for advancing the therapeutic and research applications of CRISPR-Cas technology. The potential of Cas9 in therapeutic applications, such as correcting genetic disorders and modifying gene expression, is significant, although challenges in delivery and specificity must be addressed. Overall, Cas9 represents a powerful tool for genome engineering, with broad implications for basic science, cellular engineering, and therapeutic applications.