Fig. 3.

The strategies for combining small molecule activator to control the activity of Cas9. a Recovery of intein-inactivated Cas9 via 4-HT binding. b The fusion of Cas9 and the estrogen receptor (ERT2) is sequestered in the cytoplasm, but this fusion can enter the cell nucleus via the addition of 4-HT and form a Cas9/sgRNA complex. c Without the rapamycin, the Cas9(N)-FRB-NES fragment is accumulated in the cytoplasm, while the Cas9(C)-FKBP-NLS fragment is actively introduced into the cell nucleus. d In the presence of rapamycin, Cas9(N)-FRB-NES combines with Cas9(C)-FKBP-NLS, subsequently guided into the cell nucleus by NLS. The diagram of Cas9-ssODN conjugate formation and the principle of improving HDR efficiency (e, f): e Azido-modified Cas9 binds to DBCO-modified ssODN or f DBCO-modified DNA adapter and ssODN to form Cas9-ssODN conjugates, which effectively increases the local concentration of donor ssODN near the target area