Interrogation of genome-wide, experimentally dissected gene regulatory networks reveals mechanisms underlying dynamic cellular state control

Abstract

Pooled CRISPRi-mediated silencing of >1,000 transcriptional regulators expressed in single colorectal adenocarcinoma cells, followed by single-cell RNA-seq profiling at two time points, 1D and 4D, allowed reverse engineering the underlying tumor context-specific, causal regulatory network. Furthermore, the availability of experimentally derived, highly multiplexed gene reporter assays for each regulator, as identified by this analysis, allowed accurate assessment of differential protein activity following silencing of each regulator, thus providing proof-of-concept for generating comprehensive, tissue-specific networks of transcriptional and post-translational interactions. Analysis of this causal network allowed elucidation of complex autoregulatory mechanisms that have eluded previous computational approaches and supported systematic elucidation of cooperative mechanisms, where one regulatory protein can modulate the activity of another regulatory protein, as well as transcriptional mimicry, where one regulatory protein can phenocopy others.

KSHV genome harbors both constitutive and lytically induced enhancers

“…To address the function of KSHV enhancers, we also employed CRISPR interference (CRISPRi) by directing dCas9 fused to a strong transcription repression domain (dCas9- KRAB-MeCP2) (50). SLK.219 cells were transduced with lentiviruses expressing sgRNA and dox-inducible dCas9-KRAB-MeCP2.…”

“…HEK293T cells were transfected with pLX-TRE-dCas9-KRAB-MeCP2-BSD plasmid, a gift from Andrea Califano (Addgene plasmid #140690; http://n2t.net/addgene:140690; RRID: Addgene_140690) (50) for CRISPRi or ScFv-2ERT2-VPH, a gift from Yu Wang (Addgene plasmid #120556; http://n2t.net/addgene:120556; RRID: Addgene_120556) (51), pHRdSV40- dCas9-10xGCN4_v4-P2A-BFP (Addgene plasmid # 60903; http://n2t.net/addgene:60903; RRID: Addgene_60903), a gift from Ron Vale (52) plasmids for CRISPRa with specific sgRNA ( Table S2 ) targeting KSHV enhancers and PSPAX2 lentiviral expression plasmid, a gift from Didier Trono (Addgene plasmid #12260; http://n2t.net/addgene:12260; RRID: Addgene_12260), to generate lentiviral particles. SLK.219 cells were transduced with lentiviruses supplemented with 8ug/ml polybrene.…”

“…CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in To address the function of KSHV enhancers, we also employed CRISPR interference (CRISPRi) by directing dCas9 fused to a strong transcription repression domain (dCas9-KRAB-MeCP2) (50). SLK.219 cells were transduced with lentiviruses expressing sgRNA and dox-inducible dCas9-KRAB-MeCP2.…”

Systematic evaluation of retroviral LTRs as cis-regulatory elements in mouse embryos

“…The dCas9-HA-NLS-NLS-KRAB-NLS sequence was amplified from the plasmid PB-TRE-dCas9-KRAB-MeCP2 37 (addgene 122267) using PrimeSTAR Max2× premix (Takara). A T7 promoter sequence was included in forward primer for in vitro transcription using the mMESSAGE mMACHINE T7 Ultra Transcription kit (Thermo Fisher Scientific).…”

Identification and Pharmacological Targeting of Treatment-Resistant, Stem-like Breast Cancer Cells for Combination Therapy

“…After PCR amplification, the insert was gel purified (GeneJet) and Golden-gate cloned into BsmBI-digested CROPseq-CaptureSeq-Guide-Puro-plasmid 8 . The Golden-gate cloned product was Isopropanol precipitated and large-scale electroporated into Lucigen Enduro competent cells.…”