Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model
Fig 1
Generation of the iKRAB ESC line.
(A) Schematic diagram shows the strategy of ICE to generate the iKRAB ESC line. FLAG-dCas9-KRAB was integrated into the downstream of the TRE element through homologous recombination. Dox-controlled rtTA drives the expression of fusion protein of FLAG-dCas9-KRAB. (B) Western blot analysis showing the inducible and reversible expression of FLAG-dCas9-KRAB protein at different time points after Dox addition or withdrawal. β-actin served as a loading control. A relative gray value quantification of dCas9-KRAB protein levels is below each lane of the band. (C, D) IF staining of Cas9 and FLAG in iKRAB ESC cultured with or without Dox. The scale bar represents 50 μm. Cas, CRISPR-associated; dCas9, deactivated Cas9; Dox, doxycycline; ESC, embryonic stem cell; ICE, inducible cassette exchange; IF, immunofluorescence; KRAB, Krüppel-associated box; rtTA, reverse tetracycline transcriptional activator; TRE, tetracyclin response element.