Small molecule–triggered Cas9 protein with improved genome-editing specificity

Post-translational regulation of Cas9 activity may improve the specificity of genomic targeting. A modified version of Cas9 with an insertion of a small molecule–regulated intein allows temporal control of Cas9 activity and reduces off-target activity. Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen–responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.