Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells

  1. Baolong Xia
  2. Raghuvir Viswanatha
  3. Yanhui Hu
  4. Stephanie E Mohr
  5. Norbert Perrimon  Is a corresponding author
  1. Department of Genetics, Blavatnik Institute, Harvard Medical School, United States
  2. Drosophila RNAi Screening Center, Harvard Medical School, United States
  3. Howard Hughes Medical Institute, United States
7 figures, 3 tables and 5 additional files

Figures

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 …

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 …

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 …

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 …

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 …

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 …

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…

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 …

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 …

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.
GeneHuman orthologKnown gene affecting cell fitnessReference
zldZNF485
Eaat1SLC1A3
CR44587-
Lis-1/Ptp52F*PAFAH1B1/PtprbLIS-1-overexpressing mitotic cells show a variety of spindle defectsPMID: 10722879
scylDDIT4Scyl inhibits cell growth by regulating the Tor pathwayPMID: 15545626
αTub85ETUBA1A
PoxmPax9PAX9 overexpression inhibits cancer cell proliferationPMID: 35628401
DllDLX6
LKRSDHAASSOverexpression of Aass suppresses cancer cell proliferationPMID: 31601242
scroNKX2-1NKX2-1 suppresses lung cancer progression by dampening ERK activityPMID: 34689179
CG3168SV2AOverexpression of SV2A inhibits the PI3K signaling pathwayPMID: 34277597
CG2930SLC15A1
  1. *

    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)GeneHuman orthologFunctionKnown rapamycin resistance gene
1, 1CG8468SLC16A8monocarboxylate transporter
2, 3CG5399APOD/LCN2lipocalin
5, 2CG9932ZFN462/RESTtranscription factor
4, 10CG34459/unknown
22, 13Pka-C3PRKXcatalytic subunit of PKAPMID: 15643061, 14673167, 11739804
41, 8PsNOVA1RNA splicing
43, 45CDC25CDC25A/CDC25Btyrosine phosphatasePMID: 24383842, 19276368
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Drosophila melanogaster)CG8468FlyBaseFLYB:FBgn0033913
Gene (Drosophila melanogaster)CG5399FlyBaseFLYB:FBgn0038353
Gene (Drosophila melanogaster)CG9932FlyBaseFLYB:FBgn0262160
Cell line (D. melanogaster)S2R+DRSCFLYB:FBtc0000150
Cell line (D. melanogaster)PT5DRSCFLYB:FBtc0000229
Strain, strain background (Escherichia coli)E.cloni10GF’ Electrocompetent CellsBiosearch Technologies60061–2sgRNA library construction
Strain, strain background (Escherichia coli)One Shot TOP10 Chemically Competent E. coliInvitrogenC404010
AntibodyRecombinant Anti-Insulin Receptor (phospho Y1185) antibody (Rabbit monoclonal)Abcamab623211:1000 for WB
AntibodyPhospho-Akt (Ser473) (D9E) XP antibody (Rabbit monoclonal)Cell Signaling Technology40601:1000 for WB
AntibodyAkt Rabbit Antibody (Rabbit polyclonal)Cell Signaling Technology92721:1000 for WB
AntibodyStarBright Blue 700 Goat Anti-Rabbit IgGBio-Rad120041611:2500 for WB
AntibodyStarBright Blue 520 Goat Anti-Rabbit IgGBio-Rad120058691:2500 for WB
AntibodyhFAB Rhodamine Anti-Actin Primary Antibody (synthesized, monoclonal)Bio-Rad120041631:2500 for WB
Recombinant DNA reagentpMK33-SAM plasmidThis paperCan be obtained from DRSC
Recombinant DNA reagentpLib8 plasmidThis paperU6:3-MS2 sgRNA cassette, can be obtained from DRSC
Recombinant DNA reagentpBS130 plasmidAddgene26290PhiC31 integrase
Recombinant DNA reagentpUAS-CG5399 plasmidThis paperCG5399 ORF vector, cassette, can be obtained from DRSC
Commercial assay or kitEffectene Transfection ReagentQiagen301425
Commercial assay or kitCellTiter-Glo Luminescent Cell Viability AssayPromegaG7570
Commercial assay or kitRNeasy Mini KitQiagen74104
Commercial assay or kitiScript cDNA Synthesis KitBio-Rad1708890
Chemical compound, drugMEGAscript T7 Transcription KitInvitrogenAM1334
Chemical compound, drugMethyl-β-cyclodextrinSigma-AldrichC4555
Chemical compound, drugCholesterolSigma-AldrichC3045
Chemical compound, drugCholesterolSigma-AldrichC2044
Software, algorithmGraphPad Prism 7GraphPad
Software, algorithmFlowJoFlowJo

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