Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet: (A) CRISPR/Cas9 screen workflow and screening strategy. Cas9 + Daudi B cells were transduced with the Avana sgRNA library at MOI 0.3, stimulated by multimerized CD40L at 50 ng/mL for 48 h and sorted for the 3% of cells with the lowest or highest Fas expression. (B) Scatterplots showing the statistical significance of selected hits in the screen for CD40 positive regulators. −Log10(p value) for two biological screen replicates are shown. Statistical significance was quantitated by the STARS algorithm, using one biological screen replicate for each axis. Selected CD40 positive regulator screen hits are highlighted. (C) Log2 sgRNA abundances in the indicated cell populations. sgRNA values in the input CRISPR Daudi cell library (red), in the sorted Fas low (blue) and Fas high (green) populations are shown. Mean ± SD of two input libraries and four screen replicates are shown. ***p < 0.001. (D) Scatterplots showing the −log10(p value) for two biological screen replicates. Selected CD40 negative regulator screen hits are highlighted. (E) Schematic diagram of the CD40/NF-κB pathway, highlighting known components that scored in our screens. Positive regulator Fas low screen hits with p < 0.05 (red) or negative regulator Fas high screen hit with p < 0.05 (light blue) are highlighted. See also .

Article Snippet: Avana CRISPR Lentivirus Library , Broad Institute , N/A.

Techniques: CRISPR, Transduction, Expressing

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet: (A) Log2-normlized FBXO11 sgRNA abundances within the indicated CRISPR screen cell populations. CRISPR Daudi cell library (red), in the sorted Fas low (blue) and Fas high (green) populations are shown. Mean ± SD of two input libraries and four screen replicates are shown. (B) Immunoblot analysis of whole cell extracts (WCE) from Cas9 + Daudi B cells expressing the indicated control or independent FBXO11 -targeting sgRNAs. (C) FACS analysis of PM Fas levels in Cas9 + Daudi B cells expressing the indicated control or FBXO11 targeting sgRNA and stimulated by 50 ng/mL Mega-CD40L for 48 h as shown. (D) FACS analysis of PM Fas median fluorescence intensity (MFI) as in (B) from n = 3 replicates. (E) CD40 canonical NF-κB pathway IκBα and tubulin load control levels in WCE from control or FBXO11 sgRNA expressing Daudi B cells treated with 50 ng/mL Mega-CD40L for the indicated times. The ratios of IκBα to tubulin (tub) abundances are shown beneath. (F) FACS analysis of PM CD40 MFI in Daudi B cells expressing the indicated sgRNA and stimulated by Mega-CD40L 50 ng/mL for 48 h, as indicated, using n = 3 replicates. (G) Immunoblot analysis of WCE from Cas9 + Daudi B cells expressing the indicated control or FBOX11 sgRNA from a replicate shown in (F). (H) FACS analysis of PM CD40 and Fas levels in primary spleen B cells from n = 3 wild type (WT) or FBXO11 KO mice stimulated by anti-CD40 agonist antibody (1 μg/mL) and recombinant mouse IL-4 (20 ng/mL) for 48 h. (I) Percentages of IgG1 + B cells obtained from n = 5 WT or FBXO11 KO mice stimulated for the indicated days with anti-CD40 antibody (1 μg/mL) and recombinant mouse IL-4 (20 ng/mL). All immunoblots were representative of at least n = 3 replicates. Mean + SD are shown in (D), (F), (H), and (I). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Cas9 + Daudi B cells were used for (A)–(G). See also and .

Article Snippet: Avana CRISPR Lentivirus Library , Broad Institute , N/A.

Techniques: CRISPR, Western Blot, Expressing, Control, Fluorescence, Recombinant

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet: (A) Log2-normlized CELF1 sgRNA abundances of the indicated CRISPR screen cell populations. Mean + SD of two input libraries and four screen replicates are shown. (B) FACS analysis of PM Fas levels in Cas9 + Daudi B cells expressing the indicated sgRNA and stimulated by 50 ng/mL Mega-CD40L for 48 h where indicated. (C) PM MFI Fas abundances as in (B) from n = 3 experiments. (D) Immunoblot of CELF1 or control GAPDH abundances using WCE from Cas9 + Daudi B cells expressing control or independent CELF1 targeting sgRNAs. (E) Immunoblot of CELF1, V5 epitope-tagged CEFL1, or control GAPDH abundances in WCE from single-cell Cas9 + Daudi B cell control or CELF1 KO clones with stable V5-tagged CELF1 rescue (CELF1 R ) cDNA expression. (F) FACS analysis of PM Fas abundances in Daudi B cell CELF1 KO expressing control or CELF1 R cDNAs and stimulated by Mega-CD40L (50 ng/mL) for 48 h, as indicated. (G) Immunoblot analysis of IκBα or tubulin load control abundances in WCE from Cas9 + Daudi B cells expressing control or CELF1 sgRNA and treated with 50 ng/mL Mega-CD40L for the indicated minutes (min). Ratios of IκBα/tubulin (tub) abundances are shown beneath each lane. (H) Immunoblot analysis of p100 and p52 abundances in WCE from Cas9 + Daudi B cells expressing control or CELF1 sgRNA and treated with 50 ng/mL Mega-CD40L for the indicated hours (h). Ratios of p100:p52 abundances are shown beneath each lane. The immunoblot was representative of n = 3 experiments. (I) FACS analysis of PM CD40 abundances in Cas9 + Daudi B cells expressing either control or one of two independent CELF1 sgRNAs. (J) FACS analysis of Cas9 + Daudi B cell PM CD40 MFI as in (I) from n = 3 replicates (K) Immunoblot analysis of CD40 or tubulin abundances in WCE from Cas9 + Daudi B cells expressing control or CELF1 sgRNAs. (L) Immunoblot of CD40, Poly-Ub, or tubulin abundances in WCE from Cas9 + Daudi B cells that expressed the indicated sgRNA and that were treated with control DMSO or the proteasome inhibitor bortezomib (200 nM) for 16 h. Mean + SD from n = 3 are shown in (C) and (J). **p < 0.01; ***p < 0.001. All immunoblots were representative of at least n = 3 replicates. See also .

Article Snippet: Avana CRISPR Lentivirus Library , Broad Institute , N/A.

Techniques: CRISPR, Expressing, Western Blot, Control, Clone Assay

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet: (A) Log2-normlized CRISPR screen mean + SD abundances are shown for sgRNAs targeting the gene encoding METTL14. Mean + SD of two input libraries and four screen replicates are shown. (B) FACS analysis of PM Fas abundances in Cas9 + Daudi B cells expressing control or METTL14-targeting sgRNAs and stimulated by 50 ng/mL Mega-CD40L for 48 h, as indicated. (C) Immunoblots analysis of CD40, METTL14, or control tubulin abundances in WCE from Cas9 + Daudi B cells expressing the indicated sgRNAs. (D) Immunoblots analysis of CD40, IκBα, or GAPDH abundances in Cas9 + Daudi B cells expressing the indicated sgRNAs pre-treated for 1 h with 5 μM Calbiochem IKK inhibitor VIII (IKK inh) and then treated with IKK inh together with 50 ng/mL Mega-CD40L for an additional 12 h, as indicated. (E) RT-PCR analysis of 18S-rRNA normalized CD40 mRNA levels in Cas9 + Daudi B cells expressing the indicated sgRNA. (F) RT-qPCR analysis of Daudi B cell CD40 mRNA immunopurified by control IgG versus IgG against WTAP component VIRMA, expressed as a percentage of WCE input CD40 RNA abundance. (G) RT-qPCR analysis of CD40 abundances in control IgG versus anti-m6A mRNA immunoprecipitations from Cas9 + Daudi B cells that expressed control or METTL14 sgRNAs. Immunopurified CD40 mRNA abundances are expressed as a percentage of input CD40 mRNA levels. (H) Schematic highlighting ESCRT component CD40 negative regulator screen hits. ESCRT-0, II, and III subunits hits included genes encoding HRS, VPS25,VPS36, CHMP5, and CHMP6. (I) Log2-normalized CRISPR screen CHMP5 and VPS25 sgRNA abundances. Mean + SD of two input libraries and four screen replicates are shown. (J) FACS analysis of PM Fas abundance in Cas9 + Daudi B cells with the indicated control, CHMP5 , or VPS25 sgRNAs and stimulated with 50 ng/mL Mega-CD40L for 48 h, as indicated. (K) FACS analysis of PM CD40 abundances in Cas9 + Daudi B cells with the indicated control, CHMP5 , or VPS25 sgRNAs and stimulated with 50 ng/mL Mega-CD40L for 24 h, as indicated. (L) PM MFI Fas abundances as in (K) from n = 3 experiments. (M) Immunoblot analysis of WCE from Cas9 + Daudi B cells expressing the indicated control or either of two independent CHMP5 or VPS25 targeting sgRNAs and stimulated for 24 h with 50 ng/mL Mega-CD40L, as indicated. Mean + SD from n = 3 are shown in (E)–(G), (J), and (L). *p < 0.05, **p < 0.01, ***p < 0.001. All immunoblots are representative of at least n = 3 experiments. See also and .

Article Snippet: Avana CRISPR Lentivirus Library , Broad Institute , N/A.

Techniques: CRISPR, Expressing, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet: (A) Log2-normlized CRISPR screen mean + SD abundance values for DUSP10 sgRNAs. (B) Immunoblots of DUSP10 or control GAPDH abundances in WCE from Cas9 + Daudi cells expressing control or independent DUSP10 sgRNAs. (C) FACS analysis of PM Fas levels in a representative experiment with Cas9 + Daudi cells expressing the indicated control or DUSP10 sgRNA and stimulated by 50 ng/mL Mega-CD40L for 48 h as shown. (D) FACS analysis of PM Fas MFI as in (C) from n = 3 experiments. (E) FACS analysis of PM CD40 expression in Cas9 + Daudi B cells expressing control versus DUSP10 sgRNAs. (F) Immunoblot analysis phosphorylated and total p38, ERK, and JNK MAPK abundances in WCE from Cas9 + Daudi B cells expressing the indicated sgRNAs and stimulated by 50 ng/mL Mega-CD40L for the indicated hours. Shown below are the ratios of phosphokinase to total kinase signal from a representative experiment. Ratios in unstimulated cells with control sgRNA expression were normalized to a value of 1. (G) FACS analysis of PM Fas level in Cas9 + Daudi B cells treated with 10 ng/mL TNF-α for 24 h, as indicated. (H) DUSP10 RNA-seq reads in primary human B cells stimulated with CD40L for 0, 1, or 18 h. (I) Immunoblot analysis of DUSP10 versus loading control TBP abundances from primary human B cells stimulated with 50 ng/mL Mega-CD40L for 0, 1, 4, or 18 h. Immunoblots were representative of n = 3 experiments. Mean and SD values from three independent experiments are shown in (D) and (H). *p < 0.05, ***p < 0.001. Cas9 + Daudi B cells were used in (A)–(G). See also .

Article Snippet: Avana CRISPR Lentivirus Library , Broad Institute , N/A.

Techniques: CRISPR, Western Blot, Control, Expressing, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Recovering false negatives in CRISPR fitness screens with JLOE

doi: 10.1093/nar/gkad046

Figure Lengend Snippet: False negative genes in essential protein complexes. Binary essentiality calls of essential protein complexes among colorectal cancer cell lines in the Avana data based on BAGEL derived essentiality scores. Dark blue color indicates essentiality (BF ≥ 10) and white color indicates non-essentiality (BF < 10). ( A ) Binary essentiality calls of members of 26S proteosome complex. ( B ) Binary essentiality calls of members of COP9 signalosome complex among colorectal cancer cell lines in the Avana data based on BAGEL derived essentiality scores. The relationship between screen quality ( F -measure) and the number of non- essential hits within essential protein complexes. Boxplots showing the distribution of F -measures of screens versus the number of times a non-essential call was made among members of proteosome complex ( C ) and COPS9 signalosome complex ( D ). MCM complex is an example of a CRISPR-library specific false negative. While some members of the MCM complex cannot be captured as essential in multiple colorectal screens in the Avana dataset ( E ), the complex shows more uniform essentiality in Sanger colorectal cell lines ( F ).

Article Snippet: The raw read count file of genome-wide CRISPR pooled library screens for 769 cell lines using Avana library (Broad DepMap, 2020b; Meyers et al. , 2017) (Broad Institute's DepMap Project 20Q2 release) was downloaded from the data depository at https://depmap.org/portal/ .

Techniques: Derivative Assay, CRISPR

Journal: Nucleic Acids Research

Article Title: Recovering false negatives in CRISPR fitness screens with JLOE

doi: 10.1093/nar/gkad046

Figure Lengend Snippet: JLOE approach identifies more essential genes in Avana genome-wide CRISPR-Cas9 knockout screens compared to saturation modeling approach. ( A ) Schematic description of JLOE method. ( B ) Comparison of the frequency of essential gene hits in saturation modeling versus JLOE. ( C ) Comparison of violin plots showing the distribution of the essentiality scores (Bayes Factor, BFs) of the hits in (A), in the screens where they were not observed as essential. JLOE can rescue the false negatives with a less significant increase in false positives compared to assigning binary calls using a lower BF threshold. ( D ) The number of essential gene hits using different thresholds (binary calls with BF 10, binary calls with BF 5 and mean binary call of 1 with JLOE across 100 iterations. ( E ) The number of false positive genes detected across colorectal cancer cell lines in the Avana data using same thresholds in (D). P -value annotation from t -test ind. samples with Bonferroni correction legend as follows; ns: 0.05 < P , *: 0.01 < P ≤ 0.05, **: 0.001 < P ≤ 0.01, ***: 0.0001 < P ≤ 0.001.

Article Snippet: The raw read count file of genome-wide CRISPR pooled library screens for 769 cell lines using Avana library (Broad DepMap, 2020b; Meyers et al. , 2017) (Broad Institute's DepMap Project 20Q2 release) was downloaded from the data depository at https://depmap.org/portal/ .

Techniques: Genome Wide, CRISPR, Knock-Out, Comparison

Journal: Genome Biology

Article Title: Multiple-gene targeting and mismatch tolerance can confound analysis of genome-wide pooled CRISPR screens

doi: 10.1186/s13059-019-1621-7

Figure Lengend Snippet: Single-mismatch tolerance and genetic interactions in the Achilles dataset. a Cumulative distribution of off-target delta coefficients for the set of 427 guides that have exactly one on-target and one single-mismatch off-target in the Avana library. Larger negative coefficients suggest greater off-target activity. The gray line represents the cumulative distribution of delta coefficients for 1000 guides chosen at random. b Log-fold changes (LFCs) for guides targeting seven pairs of paralog genes. Clean guides and double-target guides are shown. c Avana guide design for guides targeting LSM14A and LSM14B . d LFCs and CERES scores for guides targeting LSM14A and LSM14B . Notation: LFC (A;B): LFC for a guide with gene A as an on-target and gene B as a single-mismatch off-target. e Same as c , for YPEL1 and YPEL3 . f LFCs and CERES scores for guides targeting YPEL1 and YPEL1 . Colors indicate gene-specific cell line dependencies. Red: YPEL1 -only dependencies; black: YPEL3 -only dependencies; orange: both YPEL1 and YPEL3 dependencies; blue: no dependencies. g Same as c , for SLC25A18 and SLC25A22 . h LFCs and CERES scores for guides targeting SLC25A18 and SLC25A22 . Right panel: LFC averaged across all on-target guides targeting SLC25A18 and SLC25A22 vs LFC averaged across guides with both on-targets and off-targets. Red: SLC25A18 -dependent cell lines. Orange: SLC25A22 -dependent cell lines. Black: no dependencies

Article Snippet: Following their previous effort in identifying cancer vulnerabilities using RNAi pooled screens [ ], the Broad Institute has been performing genome-wide pooled CRISPR knockout screens across several hundreds of genomically characterized cancer cell lines [ ] using the Avana guide library [ ].

Techniques: Activity Assay