Journal: PLoS ONE

Article Title: Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

doi: 10.1371/journal.pone.0263262

Figure Lengend Snippet: (A) Viability staining of DIE cells treated with doxycycline (Doxy) concentrations (100, 250, 500 or 1000ng/ml) and for different exposure times (2, 4, 6, or 8h) to determine the optimal concentration and exposure time to induce sufficient cell death rates in DIE cells. Green circles indicate which conditions (low Doxy: 250ng/ml high Doxy: 1000ng/ml); were used for the genome-wide CRIPSR/Cas9 screen. (B) The CRISPR/Cas9 screen timeline from the time of library transfection (Day 0) to the final harvest of surviving DIE cells (Day 10). 6 days after transfection of the library, Doxycycline (Doxy) was added for 24h to induce DUX4 expression. Low Doxy: 250ng/ml; high Doxy: 1000ng/ml; early harvest: 24h after Doxy removal; late harvest: 48h after Doxy removal. (C) Volcano plot showing the enrichment of sets of guides of the low doxycycline 250ng/ml) -early harvest (24h after doxycycline removal) screen (early-low). Blue points represent guide sets that are significantly enriched (P-value ≤ 0.01), LFC ≥ 1), green points are the positive controls (DUX4, MAST1, MGAT4B), red points represent the non-target/negative control guides. (D) Chromosomal ideogram indicating the location of enriched hits in the human genome, of the low doxycycline-early harvest screen (see panel B). (E) Schematic representation of the location of a small number of false positive hits on chromosome 5 and chromosome 19. (F) Viability staining demonstrating surviving DIE-Cas9 cells (DIE cells constitutively expressing Cas9) after 250ng/ml doxycycline exposure, containing knockouts of the same genes mentioned in (E), but also DUX4, MGAT4B and MAST1. Media did not contain any selection markers (blasticidin or puromycin) to select for the presence of the rtTA3 or the DUX4 transgene. NT: Non-target controls.

Article Snippet: The Human Brunello CRISPR knockout pooled lentiviral prep library was a gift from David Root and John Doench (Broad Institute, MA, U.S.A.).

Techniques: Staining, Concentration Assay, Genome Wide, CRISPR, Transfection, Expressing, Negative Control, Selection

Journal: PLoS ONE

Article Title: Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

doi: 10.1371/journal.pone.0263262

Figure Lengend Snippet: (A) Adjusted volcano plot of screen data with low doxycycline (250ng/ml)/early harvest (24h after doxycycline removal, see ) showing the enrichment of sets of guides targeting genes not located on chromosome 5q or chromosome 19p. Blue points represent guide sets that are significantly enriched (P-value ≤ 0.01), Log2(fold change) ≥ 1), the green point is the positive control (DUX4), red points represent the non-target control guides. (B) Venn diagram showing the overlap of filtered hits between the four screens (EL: Early harvest-low Doxy, LL: Late harvest-Low doxy, EH: Early harvest-high Doxy, LH: Late harvest-High doxy), see also . (C) Viability staining showing surviving DIE cells containing single knockouts of potentials hits, identified in the CRISPR screen. Knockouts of individual genes were generated by transfection of sgRNA; 6 days later, cells were left untreated or treated with 3 different concentrations of doxycycline (100, 250 and 1000ng/ml) for and incubated for an additional 48–96 hours prior to visualizing surviving cells. Data are representative of at least three independent experiments. (D) Viability staining showing the surviving DIE-ieGFP-Cas9 cells (DIE cells expressing Cas9 constitutively and contain doxycycline-inducible eGFP) with single knockouts of mediator complex subunits. Knockouts of individual genes were generated by transfection of sgRNA; 6 days later cells were treated for doxycycline (250ng/ml) for 24h and incubated for an additional 48–96 hours prior to visualizing surviving cells. Data are representative of at least three independent experiments. (E) FACS data showing GFP-positive cells in surviving populations of DIE-ieGFP-Cas9 (expressing constitutive Cas9 and doxycycline-inducible GFP). cells containing single knockouts as indicated. Knockouts of individual genes were generated by transfection of sgRNA; 6 days later, cells were treated with doxycycline (250ng/ml) for 24h prior to FACS analysis. DIE-ieGFP-Cas9 cells comprised of 42% of eGFP-positive cells after DUX4 knockout. rtTA, MED25, MED24 and MED16 knockouts displayed a lower percentage of eGFP-expressing cells, comprising between 1.2–4% of eGFP-expressing cells. Data are representative of at least three independent experiments.

Article Snippet: The Human Brunello CRISPR knockout pooled lentiviral prep library was a gift from David Root and John Doench (Broad Institute, MA, U.S.A.).

Techniques: Positive Control, Control, Staining, CRISPR, Generated, Transfection, Incubation, Expressing, Knock-Out