Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells
- Harvard Medical School, United States
- Tsinghua University, China
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 3 supplements
A novel method for introducing highly complex DNA libraries using phiC31 recombination.
(A) phiC31 attP-attB recombination strategy. S2R+/PT5 cells containing attP sites (gold) flanking mCherry were recombined with attB donor (pLib6.4) containing attB sites (yellow) flanking U6 …
Figure 1—figure supplement 1
Copper induction is not required in PT5/MT-Cas9 cells to give maximal gene editing efficiency.
Control S2R+/PT5 cells or cells stably transfected with metallothioein-promoter-driven Cas9 (MT-Cas9) were additionally transiently transfected with Dredd-targeted sgRNA followed by editing …
Figure 1—figure supplement 2
Validation of Cas9 induction system in Drosophila S2R + cells.
(A) Cells stably expressing intein-Cas9_S219-3XFLAG (Davis et al., 2015) treated with indicated concentration of 4-HT with or without CuSO4 as indicated were subjected to anti-Flag Western blot. …
Figure 1—figure supplement 3
Design of sgRNA library vector and sgRNA PCR for next-generation sequencing.
(A) Restriction map of sgRNA library vector pLib6.4. Annealed oligos are ligated into BbsI/BpiI site as indicated. (B) Two-step PCR scheme and amplicon barcoding. Amplicons are amplified from …
Figure 2 with 1 supplement
Genome-wide CRISPR dropout screen in Drosophila S2R+ cells, results and metrics.
(A) CRISPR library is maintained in three distinct sublibrary groups as indicated, containing common controls. (B) sgRNA-level analysis of common controls in each group verify similar growth rates …
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Figure 2—source data 1
CRISPR and RNAi screen comparisons, continued.
Table containing CRISPR or RNAi Z-scores as well as RNA expression data for all targeted genes.
- https://doi.org/10.7554/eLife.36333.009
Figure 2—figure supplement 1
CRISPR and RNAi screen comparisons, continued.
(A) RNAi exhibits a stronger dependence on mRNA level than CRISPR. A rank-based binning function was applied to CRISPR or RNAi data, where every 100 genes was binned, and each biwize average Z-score …
Figure 3
Analysis of Drosophila S2R+ fitness genes.
(A) Top enriched gene ontology (GO) terms for screen hits at 5% FDR compared with top enriched GO terms for non-hits. (B) Overlap between cell-line CRISPR hits at 5% FDR and all ‘lethal’ Flybase …
Figure 4
Screens to identify genes regulating cell growth and proliferation.
(A) Schematic of selected components of the Ras/ERK/ETS and PI3K/mTor pathways and of inhibition by trametinib (‘tra’) or rapamycin (‘RAP’). (B) Experimental schematic: pathway-specific …
Additional files
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Supplementary file 1
Fitness essential gene data.
Worksheet 2 is a list of all genome-wide sgRNAs. Worksheet 1 contains computed MAGeCK (Li et al., 2014) MLE result for each independent replicate of the negative selection screen and the sgRNA-level average. Worksheet 3 contains primer sequences for cloning oligo pools. Worksheet 4 contains primers for primers for amplifying sgRNAs from cell pools (see protocol illustration in Figure 1—figure supplement 3).
- https://doi.org/10.7554/eLife.36333.012
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Supplementary file 2
Uncharacterized (‘CG’) genes and insect-specific fitness essential genes.
- https://doi.org/10.7554/eLife.36333.013
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Supplementary file 3
Context-specific fitness gene essentiality data.
Worksheet 1 contains computed MLE result of CRISPR screen conducted in each drug (tra or RAP) versus control. Worksheet 2 contains raw readcount file used to generate data.
- https://doi.org/10.7554/eLife.36333.014
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Supplementary file 4
List file for group 1 Drosophila sgRNA library.
File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.015
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Supplementary file 5
List file for group 2 Drosophila sgRNA library.
File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.016
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Supplementary file 6
List file for group 3 Drosophila sgRNA library.
File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.017
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Supplementary file 7
Readcount file for group 1, replicate 1 of Drosophila sgRNA library following transfection and outgrowth.
Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.018
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Supplementary file 8
Readcount file for group 2, replicate 1 of Drosophila sgRNA library following transfection and outgrowth.
Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.019
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Supplementary file 9
Readcount file for group 3, replicate 1 of Drosophila sgRNA library following transfection and outgrowth.
Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.020
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Supplementary file 10
Readcount file for group 1, replicate 2 of Drosophila sgRNA library following transfection and outgrowth.
Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.021
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Supplementary file 11
Readcount file for group 2, replicate 1 of Drosophila sgRNA library following transfection and outgrowth.
Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.022
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Supplementary file 12
Readcount file for group 3, replicate 1 of Drosophila sgRNA library following transfection and outgrowth.
Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.023
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Supplementary file 13
Readcount file for average replicates 1 and 2, group 1.
Readcounts were internally normalized to a median value of 10000 prior to computing the average. Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.024
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Supplementary file 14
Readcount file for average replicates 1 and 2, group 2.
Readcounts were internally normalized to a median value of 10000 prior to computing the average. Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.025
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Supplementary file 15
Readcount file for average replicates 1 and 2, group 3.
Readcounts were internally normalized to a median value of 10000 prior to computing the average. Column 1 provides internal ID number compatible with Supplementary file 1. Column 2 provides targeted gene ID. Column 3, ‘REF’, provides readcount file from plasmid pool. Column 4 provides readcount following transfection and outgrowth. File is compatible with MAGeCK (Li et al., 2014).
- https://doi.org/10.7554/eLife.36333.026
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Transparent reporting form
- https://doi.org/10.7554/eLife.36333.027
