Figure 4.

Validation of GCLC inducible sgRNA systems and in vivo knockout efficacy. (A, B) MV4;11.FLuc cells were stably transduced with Cas9 and indicated inducible GCLC sgRNAs. After 4 days of induction with 1 µg/ml Doxycycline (Dox), GCLC edit efficiency was analyzed by Sanger sequencing and ICE analysis (A) and GCLC protein level was detected using capillary Wes analysis (B). (C, D) MV4;11.FLuc cells transduced with sgNC, sgGCLC1, or sgGCLC3 were induced with 1 µg/ml Doxycycline. Cell growth was measured using CTG assay on day 6 (C) and colony formation was detected on day 8 (D). (E) NSG mice were intravenously injected with either MV4;11.FLuc.sgNC, MV4;11.FLuc.sgGCLC1, or MV4;11.FLuc.sgGCLC3 cells (5 × 10⁶ cells/mouse), followed by feeding control or Dox diet starting from 18 days after implantation. Mouse BM cells were collected 5 or 7 days after dosing for PD studies. (F, G) Human CD45⁺ cell populations in BM samples from mice implanted with MV4;11.FLuc.sgGCLC1 (F) or MV4;11.FLuc.sgGCLC3 (G) were detected by flow cytometry. (H, I) Human GCLC gene was detected by PCR. (J) Genomic sequencing was performed to evaluate GCLC knockout efficiency in BM samples from mice transplanted with MV4;11.FLuc.sgNC or MV4;11.FLuc.sgGCLC3 cells and fed with control or Dox diet.