(A) Schematic of the SAM complex for inducible transcriptional activation. dCas9-VP64 and MCP-p65-HSF1 were driven by an inducible metallothionein promoter. dCas9-VP64 and MCP-p65-HSF1 were …
Full source data for Figure 1.
(A) Schematic of CRISPR activation screen (See methods). (B) Two replicates of genome-wide CRISPR activation screen. Data were analyzed by MAGeCK-RRA, a smaller RRA score indicates a stronger …
Full source data for Figure 2.
(A) Schematic of pooled library cells generation. Synergistic activation mediator (SAM) complex was inserted into attP sites containing S2R+ cells to generate SAM cells. The pooled attB sites …
Full source data for Figure 2—figure supplement 1.
(A) Schematic of dual-sgRNA library design. Two different sgRNAs targeting the same gene promoter region within 500 bp upstream of the transcriptional start site (TSS). Two sgRNAs were driven by …
Full source data for Figure 2—figure supplement 2.
(A) Genome-wide cell fitness screen. Each dot represents a gene. Significantly depleted genes (false-discovery rate (FDR)<0.05) after proliferation in DMSO for 3 weeks are highlighted. (B) Counts of …
Full source data for Figure 2—figure supplement 3.
(A) Phospho-S6 levels in cells expressing dual-sgRNA vectors in the presence of 1 nM rapamycin. Western blot signals are quantitatively analyzed by ImageJ. Three biological replicates are shown as …
Full source data for Figure 3.
(A) Phospho-Akt in CG5399-overexpressing cells following Pi3K92E knockdown. Two nonoverlapping double-stranded RNAs (dsRNAs) targeting Pi3K92E were used. (B) Phospho-Akt and phospho-S6 in CG5399-over…
Full source data for Figure 3—figure supplement 1.
(A) Phospho-InR, phospho-Akt, and phospho-S6 in CG5399-overexpressing cells treated with methyl-beta-cyclodextrin (MβCD) at different concentrations. (B) Phospho-InR, phospho-Akt, and phospho-S6 in …
Full source data for Figure 4.
(A) Structure prediction of CG5399 by AlphaFold. Different side views are shown. (B) Interaction between CG5399 and cholesterol by molecular docking. Cholesterol is predicted to be inserted into the …
Full source data for Figure 4—figure supplement 1.
Gene | Human ortholog | Known gene affecting cell fitness | Reference |
---|---|---|---|
zld | ZNF485 | ||
Eaat1 | SLC1A3 | ||
CR44587 | - | ||
Lis-1/Ptp52F* | PAFAH1B1/Ptprb | LIS-1-overexpressing mitotic cells show a variety of spindle defects | PMID: 10722879 |
scyl | DDIT4 | Scyl inhibits cell growth by regulating the Tor pathway | PMID: 15545626 |
αTub85E | TUBA1A | ||
Poxm | Pax9 | PAX9 overexpression inhibits cancer cell proliferation | PMID: 35628401 |
Dll | DLX6 | ||
LKRSDH | AASS | Overexpression of Aass suppresses cancer cell proliferation | PMID: 31601242 |
scro | NKX2-1 | NKX2-1 suppresses lung cancer progression by dampening ERK activity | PMID: 34689179 |
CG3168 | SV2A | Overexpression of SV2A inhibits the PI3K signaling pathway | PMID: 34277597 |
CG2930 | SLC15A1 |
Lis-1 and Ptp52F form divergent gene pair ~500 bp apart.
Rank (Rep 1, Rep 2) | Gene | Human ortholog | Function | Known rapamycin resistance gene |
---|---|---|---|---|
1, 1 | CG8468 | SLC16A8 | monocarboxylate transporter | |
2, 3 | CG5399 | APOD/LCN2 | lipocalin | |
5, 2 | CG9932 | ZFN462/REST | transcription factor | |
4, 10 | CG34459 | / | unknown | |
22, 13 | Pka-C3 | PRKX | catalytic subunit of PKA | PMID: 15643061, 14673167, 11739804 |
41, 8 | Ps | NOVA1 | RNA splicing | |
43, 45 | CDC25 | CDC25A/CDC25B | tyrosine phosphatase | PMID: 24383842, 19276368 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Drosophila melanogaster) | CG8468 | FlyBase | FLYB:FBgn0033913 | |
Gene (Drosophila melanogaster) | CG5399 | FlyBase | FLYB:FBgn0038353 | |
Gene (Drosophila melanogaster) | CG9932 | FlyBase | FLYB:FBgn0262160 | |
Cell line (D. melanogaster) | S2R+ | DRSC | FLYB:FBtc0000150 | |
Cell line (D. melanogaster) | PT5 | DRSC | FLYB:FBtc0000229 | |
Strain, strain background (Escherichia coli) | E.cloni10GF’ Electrocompetent Cells | Biosearch Technologies | 60061–2 | sgRNA library construction |
Strain, strain background (Escherichia coli) | One Shot TOP10 Chemically Competent E. coli | Invitrogen | C404010 | |
Antibody | Recombinant Anti-Insulin Receptor (phospho Y1185) antibody (Rabbit monoclonal) | Abcam | ab62321 | 1:1000 for WB |
Antibody | Phospho-Akt (Ser473) (D9E) XP antibody (Rabbit monoclonal) | Cell Signaling Technology | 4060 | 1:1000 for WB |
Antibody | Akt Rabbit Antibody (Rabbit polyclonal) | Cell Signaling Technology | 9272 | 1:1000 for WB |
Antibody | StarBright Blue 700 Goat Anti-Rabbit IgG | Bio-Rad | 12004161 | 1:2500 for WB |
Antibody | StarBright Blue 520 Goat Anti-Rabbit IgG | Bio-Rad | 12005869 | 1:2500 for WB |
Antibody | hFAB Rhodamine Anti-Actin Primary Antibody (synthesized, monoclonal) | Bio-Rad | 12004163 | 1:2500 for WB |
Recombinant DNA reagent | pMK33-SAM plasmid | This paper | Can be obtained from DRSC | |
Recombinant DNA reagent | pLib8 plasmid | This paper | U6:3-MS2 sgRNA cassette, can be obtained from DRSC | |
Recombinant DNA reagent | pBS130 plasmid | Addgene | 26290 | PhiC31 integrase |
Recombinant DNA reagent | pUAS-CG5399 plasmid | This paper | CG5399 ORF vector, cassette, can be obtained from DRSC | |
Commercial assay or kit | Effectene Transfection Reagent | Qiagen | 301425 | |
Commercial assay or kit | CellTiter-Glo Luminescent Cell Viability Assay | Promega | G7570 | |
Commercial assay or kit | RNeasy Mini Kit | Qiagen | 74104 | |
Commercial assay or kit | iScript cDNA Synthesis Kit | Bio-Rad | 1708890 | |
Chemical compound, drug | MEGAscript T7 Transcription Kit | Invitrogen | AM1334 | |
Chemical compound, drug | Methyl-β-cyclodextrin | Sigma-Aldrich | C4555 | |
Chemical compound, drug | Cholesterol | Sigma-Aldrich | C3045 | |
Chemical compound, drug | Cholesterol | Sigma-Aldrich | C2044 | |
Software, algorithm | GraphPad Prism 7 | GraphPad | ||
Software, algorithm | FlowJo | FlowJo |
sgRNA vectors used in this study.
PCR primers used in this study.
dsRNAs used in this study.
The original files of the full raw unedited blots and figures with the uncropped blots with relevant bands labeled in this study.