Figure 1.

CRISPRoff potentially and specifically represses MGMT in GBM cells. (A) Schematic of CRISPRoff mRNA and sgRNA delivery using electroporation into GBM cells, leading to gene repression through DNMT3A/3L and KRAB domains. (B) Genomic locus of the MGMT promoter with protospacer locations of the 3 independent sgRNAs targeting the CpG island, as well as 4 CpG sites significantly associated with a hypermutated subtype of glioma with improved survival reported in.³¹ (C) RT-qPCR of MGMT mRNA normalized to RPLP0 housekeeping gene, in polyclonal and monoclonal LN18 cells at the indicated number of days following delivery of CRISPRoff and the indicated sgRNA. Primer Set K and O represent 2 independent qPCR primer pairs from (Kreth et al. 2011) and Origene, respectively. (D) RT-qPCR of MGMT mRNA as in (C) for LN18 cells delivered with a pool of all 3 sgRNAs against MGMT. (E) RT-qPCR of MGMT mRNA in polyclonal and monoclonal T98G cells following delivery of CRISPRoff and indicated sgRNA(s). (F) Western blot against MGMT protein in polyclonal and monoclonal LN18 cells at the indicated number of days following delivery of CRISPRoff and the indicated sgRNA. (G) Western blot against MGMT protein as in (F) for LN18 cells delivered with a pool of all 3 sgRNAs against MGMT. (H) Western blot against MGMT protein as in (F) for T98G cells. (I) Targeted bisulfite sequencing of the MGMT promoter, assessing 17 CpG sites in LN18 monoclonal cells following delivery of CRISPRoff and the indicated sgRNA. (J) RNA-seq volcano plots comparing CRISPRoff targeting of MGMT promoter across 2 independent sgRNAs (sgMGMT-1B, -3B) in intermediate (40 – 64d) and late (134–209d) time points.