A study published in *Nature Biotechnology* (2013) investigates the off-target DNA cleavage specificity of RNA-programmed Cas9 nucleases. The researchers used in vitro selection and high-throughput sequencing to analyze the cleavage activity of eight Cas9:guide RNA (sgRNA) complexes against 10^12 potential off-target DNA sequences. They identified five off-target sites in the human genome that were confirmed to be cleaved in HEK293T cells when specific Cas9:sgRNA complexes were expressed. The results show that Cas9:sgRNA specificity extends beyond a 7-12 base pair seed sequence, contradicting previous models. The study also reveals a trade-off between activity and specificity: shorter, less-active sgRNAs are more specific than longer, more-active ones. High concentrations of Cas9:sgRNA can cleave off-target sites with mutations near or within the PAM, which are not cleaved when enzyme concentrations are limiting. The study compares the specificity of Cas9:sgRNA with zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), highlighting the advantages of RNA-guided Cas9 in genome editing. The research demonstrates that Cas9:sgRNA can cleave DNA at sites complementary to a 20-base pair guide RNA, with a PAM sequence (NGG) at the 3' end. Previous studies suggested that perfect complementarity between guide RNA and target DNA is required in the 7-12 base pairs adjacent to the PAM, while mismatches are tolerated at the non-PAM end. However, the current study shows that the entire 20-base pair target site and two-base pair PAM contribute to Cas9:sgRNA specificity, contradicting earlier findings. The study also reveals that the architecture of sgRNA affects off-target cleavage activity. Shorter, less-active sgRNA v1.0 constructs are less tolerant of mutations than longer, more-active sgRNA v2.1 constructs. The results indicate that different guide RNA architectures can significantly influence Cas9 activity and specificity in cells. The study confirms that Cas9:sgRNA complexes can cleave off-target sites in human cells, some of which contain four mutations relative to the on-target site. These findings provide insights into RNA-programmed Cas9 specificity and highlight the importance of sgRNA architecture in DNA-cleavage specificity. The study also suggests that the principles revealed may apply to Cas9-based effectors engineered for functions beyond DNA cleavage.A study published in *Nature Biotechnology* (2013) investigates the off-target DNA cleavage specificity of RNA-programmed Cas9 nucleases. The researchers used in vitro selection and high-throughput sequencing to analyze the cleavage activity of eight Cas9:guide RNA (sgRNA) complexes against 10^12 potential off-target DNA sequences. They identified five off-target sites in the human genome that were confirmed to be cleaved in HEK293T cells when specific Cas9:sgRNA complexes were expressed. The results show that Cas9:sgRNA specificity extends beyond a 7-12 base pair seed sequence, contradicting previous models. The study also reveals a trade-off between activity and specificity: shorter, less-active sgRNAs are more specific than longer, more-active ones. High concentrations of Cas9:sgRNA can cleave off-target sites with mutations near or within the PAM, which are not cleaved when enzyme concentrations are limiting. The study compares the specificity of Cas9:sgRNA with zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), highlighting the advantages of RNA-guided Cas9 in genome editing. The research demonstrates that Cas9:sgRNA can cleave DNA at sites complementary to a 20-base pair guide RNA, with a PAM sequence (NGG) at the 3' end. Previous studies suggested that perfect complementarity between guide RNA and target DNA is required in the 7-12 base pairs adjacent to the PAM, while mismatches are tolerated at the non-PAM end. However, the current study shows that the entire 20-base pair target site and two-base pair PAM contribute to Cas9:sgRNA specificity, contradicting earlier findings. The study also reveals that the architecture of sgRNA affects off-target cleavage activity. Shorter, less-active sgRNA v1.0 constructs are less tolerant of mutations than longer, more-active sgRNA v2.1 constructs. The results indicate that different guide RNA architectures can significantly influence Cas9 activity and specificity in cells. The study confirms that Cas9:sgRNA complexes can cleave off-target sites in human cells, some of which contain four mutations relative to the on-target site. These findings provide insights into RNA-programmed Cas9 specificity and highlight the importance of sgRNA architecture in DNA-cleavage specificity. The study also suggests that the principles revealed may apply to Cas9-based effectors engineered for functions beyond DNA cleavage.