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. 2016 Jul 25;44(19):e149. doi: 10.1093/nar/gkw660

Figure 1.

Figure 1.

A sensitive and efficient inducible CRISPR/Cas9 system. (A) Schematic representation of the Lenti-iCas9-neo plasmid. LTR, long terminal repeat; PSI, retroviral Ψ packaging element; RRE, Rev response element; TRE2, TRE2 promoter; P2A, a 2A self-cleavage peptide from porcine teschovirus-1; Ubc, Ubiquitin C promoter; rtTA3, reverse tetracycline-controlled transactivator 3; IRES, internal ribosome entry site; Neo, neomycin resistance. (B) The indicated cells were transduced with Lenti-iCas9-neo and LentiGuide carrying the indicated sgRNA. Cells were then treated with or without 1 μg/ml doxycycline (DOX) for 72 h and analyzed by western blots. (C) Overview of the FACS sorting strategy. (D) Unsorted and sorted HeLa/iCas9 cells were treated with or without 1 μg/ml DOX for 24 h and GFP expression was analyzed by FACS. (E–G) SKBR3 (E), Hela (F) and MCF10A (G) iCas9 cells before or after FACS sorting were transduced with LentiGuide carrying control or KDM5C sgRNA. Cells were then treated with or without 1 μg/ml DOX for 72 h and analyzed by western blots.