Small Molecule–Triggered Cas9 Protein with Reduced Off-Target Cleavage

The RNA-guided endonuclease Cas9 enables simple and efficient genome editing. We have developed Cas9 nucleases that are activated by the cell-permeable small molecule 4-hydroxytamoxifen (4-HT) by inserting a 4-HT-responsive intein at specific positions in Cas9. These intein-Cas9s enable small-molecule control of genome editing, with Cas9 activity being controlled at the post-translational level. In human cells, these conditionally active intein-Cas9s demonstrate improved genome-editing specificity over wild-type Cas9.