Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen
Fig 1
Creation and validation of the DIE cell line.
(A) Constructs expressed in the DIE cell line to induce expression of DUX4. i) Top: Schematic representation of the rtTA3 construct (integrated on chromosome 5), constitutively expressing rtTA3 (under control of the CMV promoter) with selection marker blasticidin (BlastR, SV40 promoter). Bottom: LSL (LoxP-dsRed-stop-LoxP)-DUX4 cassette under control of TRE, tetracycline-responsive element, inducible by tetracycline-derivative doxycycline, together with selection marker puromycin (PuroR, PGK promoter), integrated on chromosome 19. dsRed but not DUX4 can be induced, as DUX4 is out of frame with dsRed. Action of CRE recombinase removed dsRed between the LoxP sites, enabling doxycycline-inducible DUX4 expression. ii) In the absence of doxycycline, rtTA3 does not bind to the TRE; addition of doxycycline results in rtTA3-TRE binding and subsequent transcription of DUX4. (B) Phase-contrast images of DIE cells without doxycycline and 24h after doxycycline exposure (1000ng/ml). (C) Schematic representation of transgene integration sites within human genome, by TLA analysis. The inducible DUX4 cassette maps to the p-arm of chromosome 19, and the rtTA3 transgene maps to the end of the q-arm of chromosome 5. (D) Analysis of DUX4 expression by qRT-PCR and western blot in KBM7, DIE and DIE-KO cells (DIE cells with knockout for DUX4) with or without doxycycline addition (500ng/ml and 1000ng/ml in DIE cells, 1000ng/ml in KBM7 and DIE KO cells), as detected by qRT-PCR (top panel) and western blot analysis (bottom panel). β -actin was used as a loading. Fold induction was calculated by 2^-(ddCT) of untreated or doxycycline-treated cells normalized by HPRT expression. Statistical significance was determined by ANOVA analysis. (E) Phase-contrast images of DIE cells which were transduced with either Cas9 protein, or Cas9 protein with DUX4 sgRNA prior to treatment with doxycycline (1000ng/ml) for at least 24h. Dead cells were removed by a DPBS wash to expose the surviving population. (F) Induction of mRNA expression of known downstream targets of DUX4 upon doxycycline treatment (1000ng/ml) in KBM7, DIE and DIE-KO cells, as measured by qRT-PCR. Fold induction was calculated by 2^-(ddCT) of uninduced or doxycycline-induced cells normalized by HPRT expression. Statistical significance was determined by ANOVA analysis.