The study by Pattanayak et al. investigates the off-target DNA cleavage specificity of the RNA-programmable Cas9 endonuclease. Using in vitro selection and high-throughput sequencing, the researchers determined the propensity of eight Cas9:guide RNA complexes to cleave 10×12 potential off-target DNA sequences. The results revealed that Cas9:guide RNA specificity extends beyond a 7- to 12-base pair seed sequence, with the entire 20-base pair target site and the two-base pair PAM contributing to cleavage specificity. The study also found that increasing Cas9:guide RNA concentrations can decrease DNA-cleaving specificity, and that shorter, less active guide RNAs are more specific than longer, more active ones. In vitro selection results were validated in human cells, where several off-target substrates identified in the selection process were confirmed to be cleaved in HEK293T cells. These findings provide insights into the specificity of RNA-programmed Cas9 and suggest that guide RNA architecture can significantly influence both activity and specificity in cells.The study by Pattanayak et al. investigates the off-target DNA cleavage specificity of the RNA-programmable Cas9 endonuclease. Using in vitro selection and high-throughput sequencing, the researchers determined the propensity of eight Cas9:guide RNA complexes to cleave 10×12 potential off-target DNA sequences. The results revealed that Cas9:guide RNA specificity extends beyond a 7- to 12-base pair seed sequence, with the entire 20-base pair target site and the two-base pair PAM contributing to cleavage specificity. The study also found that increasing Cas9:guide RNA concentrations can decrease DNA-cleaving specificity, and that shorter, less active guide RNAs are more specific than longer, more active ones. In vitro selection results were validated in human cells, where several off-target substrates identified in the selection process were confirmed to be cleaved in HEK293T cells. These findings provide insights into the specificity of RNA-programmed Cas9 and suggest that guide RNA architecture can significantly influence both activity and specificity in cells.