Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells
- Department of Genetics, Blavatnik Institute, Harvard Medical School, United States
- Drosophila RNAi Screening Center, Harvard Medical School, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1
Inducible transcriptional activation by the synergistic activation mediator (SAM) complex in Drosophila cells.
(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 …
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Figure 1—source data 1
Full source data for Figure 1.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig1-data1-v1.zip
Figure 2 with 3 supplements
Genome-wide CRISPR activation screen for rapamycin resistance genes.
(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 …
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Figure 2—source data 1
Full source data for Figure 2.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig2-data1-v1.zip
Figure 2—figure supplement 1
Pooled CRISPR activation screen with a focused library.
(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 …
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Figure 2—figure supplement 1—source data 1
Full source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
Design of genome-wide dual-sgRNA library.
(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 …
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Figure 2—figure supplement 2—source data 1
Full source data for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig2-figsupp2-data1-v1.zip
Figure 2—figure supplement 3
Genome-wide cell fitness screen and rapamycin screen.
(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 …
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Figure 2—figure supplement 3—source data 1
Full source data for Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig2-figsupp3-data1-v1.zip
Figure 3 with 1 supplement
CG5399 overexpression activates RTK-Akt-mTOR signaling.
(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 …
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Figure 3—source data 1
Full source data for Figure 3.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig3-data1-v1.zip
Figure 3—figure supplement 1
CG5399 overexpression activates Akt-mTOR through RTK/PI3K.
(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…
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Figure 3—figure supplement 1—source data 1
Full source data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig3-figsupp1-data1-v1.zip
Figure 4 with 1 supplement
Activation of InR-Akt-mTOR signaling by CG5399 overexpression requires cholesterol and clathrin-coated pits at the membrane.
(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 …
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Figure 4—source data 1
Full source data for Figure 4.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig4-data1-v1.zip
Figure 4—figure supplement 1
CG5399 interacts with cholesterol by molecular docking.
(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 …
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Figure 4—figure supplement 1—source data 1
Full source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/85542/elife-85542-fig4-figsupp1-data1-v1.zip
Author response image 1
Reads of CG5399 sgRNAs in the initial population (CG5399 sgRNAs are in red, other sgRNAs are in gray).
Author response image 2
Reads of CG8468 sgRNAs in the initial population (CG8468 sgRNAs are in red, other sgRNAs are in gray).
Author response image 3
Reads of CG9932 sgRNAs in the initial population (CG9932 sgRNAs are in red, other sgRNAs are in gray).
Tables
Table 1
Significantly depleted genes in genome-wide fitness screen.
| 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 |
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*
Lis-1 and Ptp52F form divergent gene pair ~500 bp apart.
Table 2
Overlapping genes of top-ranked 50 hits from two genome-wide screen replicates.
| 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 |
Key resources table
| 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 |
Additional files
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Supplementary file 1
sgRNA vectors used in this study.
- https://cdn.elifesciences.org/articles/85542/elife-85542-supp1-v1.docx
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Supplementary file 2
PCR primers used in this study.
- https://cdn.elifesciences.org/articles/85542/elife-85542-supp2-v1.docx
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Supplementary file 3
dsRNAs used in this study.
- https://cdn.elifesciences.org/articles/85542/elife-85542-supp3-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85542/elife-85542-mdarchecklist1-v1.docx
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Source data 1
The original files of the full raw unedited blots and figures with the uncropped blots with relevant bands labeled in this study.
- https://cdn.elifesciences.org/articles/85542/elife-85542-data1-v1.zip
