Figure 2.

Small molecule inducible activation and split systems to control sgRNA or Cas9 expression

(A) The doxycycline-inducible sgRNA expression system⁷¹ has a Tet repressor that keeps the sgRNA from being translated, so that Cas9 is not functional. However, once doxycycline is added, the repressors will no longer bind to the Tet-responsive sequence (TetO) and sgRNA will be expressed to use with Cas9. (B) The rapamycin-inducible Cas9⁷² is split into two lobes, an N terminus lobe that has the FRB component and a nuclear export signal (NES), and the C terminus lobe that has FKBP and a nuclear localization signal (NLS). Without rapamycin, only the C terminus Cas9 goes into the nucleus and is not functional. With the addition of rapamycin, the lobes are fused via FRB and FKBP binding and the functional Cas9 is shuttled to the nucleus for activity. (C) The 4-hydroxytamoxifen (4-OHT)-responsive split Cas9⁷³ is sequestered in the cytoplasm and has multiple ligand domains of the estrogen receptor (ERT). Addition of 4-OHT releases the Cas9 fragments from a heat shock protein (Hsp90) and translocates the reconstituted Cas9 to the nucleus for genome editing.