The study investigates how the CRISPR-associated enzyme Cas9, guided by RNA, identifies and cleaves specific DNA sequences. Cas9:guide RNA complexes are effective in targeting DNA in bacteria, animals, and plants. The research uses single-molecule and bulk biochemical experiments to determine how Cas9:RNA locates and cleaves DNA. It shows that both DNA binding and cleavage by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission. RNA-mediated adaptive immune systems in bacteria and archaea rely on CRISPRs and CRISPR-associated (Cas) proteins to provide protection from invading viruses and plasmids. Bacteria with CRISPR-Cas loci respond to viral and plasmid challenges by integrating short fragments of the foreign nucleic acid into the host chromosome. Transcription of the CRISPR array followed by enzymatic processing yields short CRISPR RNAs (crRNAs) that direct Cas protein-mediated cleavage of complementary target sequences within invading viral or plasmid DNA. In Type II CRISPR-Cas systems, Cas9 functions as an RNA-guided endonuclease that uses a dual-guide RNA consisting of crRNA and trans-activating crRNA (tracrRNA) for target recognition and cleavage by a mechanism involving two nuclease active sites that together generate dsDNA breaks. RNA-programmed Cas9 can be a versatile tool for genome engineering in multiple cell types and organisms. Guided by either a natural dual-RNA complex or a chimeric single-guide RNA, Cas9 generates site-specific DSBs that are repaired either by non-homologous end joining or homologous recombination. In addition, catalytically inactive Cas9 alone or fused to transcriptional activator or repressor domains can be used to alter transcription levels at sites targeted by guide RNAs. Despite the ease in applying this technology, the fundamental mechanism that enables Cas9:RNA to locate specific 20 base-pair DNA targets within the vast sequence space of genomes remains unknown. The study uses single-tethered DNA curtain assays and total internal reflection fluorescence microscopy (TIRFM) to visualize the binding site distribution of single Cas9:RNA molecules on λ- DNA substrates. The results show that DNA targeting by Cas9:RNA is faithfully recapitulated in the DNA curtain assays. The study also confirms that theThe study investigates how the CRISPR-associated enzyme Cas9, guided by RNA, identifies and cleaves specific DNA sequences. Cas9:guide RNA complexes are effective in targeting DNA in bacteria, animals, and plants. The research uses single-molecule and bulk biochemical experiments to determine how Cas9:RNA locates and cleaves DNA. It shows that both DNA binding and cleavage by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission. RNA-mediated adaptive immune systems in bacteria and archaea rely on CRISPRs and CRISPR-associated (Cas) proteins to provide protection from invading viruses and plasmids. Bacteria with CRISPR-Cas loci respond to viral and plasmid challenges by integrating short fragments of the foreign nucleic acid into the host chromosome. Transcription of the CRISPR array followed by enzymatic processing yields short CRISPR RNAs (crRNAs) that direct Cas protein-mediated cleavage of complementary target sequences within invading viral or plasmid DNA. In Type II CRISPR-Cas systems, Cas9 functions as an RNA-guided endonuclease that uses a dual-guide RNA consisting of crRNA and trans-activating crRNA (tracrRNA) for target recognition and cleavage by a mechanism involving two nuclease active sites that together generate dsDNA breaks. RNA-programmed Cas9 can be a versatile tool for genome engineering in multiple cell types and organisms. Guided by either a natural dual-RNA complex or a chimeric single-guide RNA, Cas9 generates site-specific DSBs that are repaired either by non-homologous end joining or homologous recombination. In addition, catalytically inactive Cas9 alone or fused to transcriptional activator or repressor domains can be used to alter transcription levels at sites targeted by guide RNAs. Despite the ease in applying this technology, the fundamental mechanism that enables Cas9:RNA to locate specific 20 base-pair DNA targets within the vast sequence space of genomes remains unknown. The study uses single-tethered DNA curtain assays and total internal reflection fluorescence microscopy (TIRFM) to visualize the binding site distribution of single Cas9:RNA molecules on λ- DNA substrates. The results show that DNA targeting by Cas9:RNA is faithfully recapitulated in the DNA curtain assays. The study also confirms that the