brunello lentiviral library  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: Genome-wide CRISPR knockout screens for proliferation and cisplatin sensitization of HeLa WT and EXO1-knockout cells. ( A ) Overview of the CRISPR knockout screens to identify genes that are required for proliferation and cisplatin sensitivity of WT and EXO1-knockout HeLa cells. Created in BioRender. Moldovan, G. (2026); https://BioRender.com/c77sziu . ( B ) The cellular survival of WT and EXO1-knockout HeLa cells at each splitting time. Survival was calculated by dividing the number of live cells in the cisplatin-treatment population to the control (no treatment) population.

Article Snippet: For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ].

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

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: Analyses of the cisplatin sensitivity CRISPR screens in WT and EXO1-knockout cells. ( A ) Functional annotation clustering of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in WT HeLa cells, using Gene Ontology and Uniprot terms. ( B ) Table showing the biological processes and corresponding genes from the Gene Ontology pathway analysis of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in WT HeLa cells. GO_BP terms with negative logP >1 are presented. ( C, D ) Functional annotation clustering of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in HeLa-EXO1 KO#1 ( C ) and HeLa-EXO1 KO#3 ( D ) cells using Gene Ontology and Uniprot terms. ( E ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in the two EXO1-knockout cell lines. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( F ) The number of common genes within the top cisplatin sensitivity hits in the two EXO1-knockout cell lines compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( G ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in WT and EXO1 KO#1 cells. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( H ) The number of common genes within the top cisplatin sensitivity hits in WT and EXO1 KO#1 cells compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( I ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in WT and EXO1 KO#3 cells. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( J ) The number of common genes within the top cisplatin sensitivity hits in WT and EXO1 KO#3 cells compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis.

Article Snippet: For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ].

Techniques: CRISPR, Knock-Out, Functional Assay

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: Analyses of the EXO1 synthetic lethality CRISPR screens. ( A ) Diagram showing the overlap of identical genes within the top synthetic lethality hits in the two EXO1-knockout cell lines. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( B ) The number of common genes within the top synthetic lethality hits in the two EXO1-knockout cell lines compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( C ) Functional annotation clustering of the common genes within the top synthetic lethality hits with MAGeCK score lower than 0.015 in the two EXO1-knockout cell lines, using Gene Ontology and Uniprot terms. ( D ) Diagram showing the overlap of identical genes within the top synthetic lethality hits in EXO1 KO#1 cells (compared to WT) and the control comparison to EXO1 KO#3 . The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( E ) The number of common genes within the top synthetic lethality hits in EXO1 KO#1 cells (compared to WT) and the control comparison to EXO1 KO#3 . The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis.

Article Snippet: For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ].

Techniques: CRISPR, Knock-Out, Functional Assay, Control, Comparison

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: Co-depletion of EXO1 and CHAF1A reduces cellular viability. ( A, C, E ). Volcano plots showing the results of genome-wide CRISPR knockout screens to identify EXO1 synthetic lethality interactions. Genes targeted by the library are presented based on their impact on the viability of EXO1 KO#1 compared to WT cells ( A ), EXO1 KO#3 compared to WT cells ( C ), and as control, EXO1 KO#1 compared to EXO1 KO#3 cells ( E ). Genes are plotted by the −log 10 of their respective negative and positive P -values and associated log 2 Fold Change values. The hit chosen for validation, namely CHAF1A, is indicated. ( B, D, F ) Scatterplots showing the results of genome-wide CRISPR knockout screens to identify EXO1 synthetic lethality interactions. Genes targeted by the library are plotted based on their impact on the viability of EXO1 KO#1 compared to WT cells ( B ), EXO1 KO#3 compared to WT cells ( D ), and as control, EXO1 KO#1 compared to EXO1 KO#3 cells ( F ). The hit chosen for validation, namely CHAF1A, is indicated. ( G ) Table showing the ranks in the synthetic lethality screens, and the biological roles of CHAF1A. ( H, I ) Clonogenic survival assays showing that siRNA ( H ) and sgRNA ( I ) depletion of CHAF1A reduces the viability of EXO1-knockout cells compared to WT HeLa cells. Clonogenic survival is presented normalized to WT control cells. The average of three independent experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance ( t -test unpaired).

Article Snippet: For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ].

Techniques: Genome Wide, CRISPR, Knock-Out, Control, Biomarker Discovery

Journal: bioRxiv

Article Title: High-throughput Genome Wide CRISPR Knock Out mechanical sort identifies genes driving metastatic cancer cell softening

doi: 10.64898/2026.02.12.705447

Figure Lengend Snippet: Cancer cells expressing Cas9 are transduced with a lentiviral library containing sgRNAs across the entire protein-coding genome at a low MOI to allow for single gene knockouts. Antibiotic selection ensures only cells with gene knockouts are included in mechanical screen. Next, the library of cells with single gene knockouts are processed with a microfluidic stiffness-based cell sorting device into 5 mechanical subsets. The inset shows a side and top view of the ridged region of the device where cells must deform underneath each diagonal ridge. Softer cells deform easily underneath the ridges and follow hydrodynamic streamlines to provide a slight negative deflection towards outlets 1 and 2. Stiffer cells resist deformation and are deflected along the diagonal ridge resulting in trajectories towards outlets 4 and 5. Genomic DNA is harvested from each mechanical subset, the sgRNA regions are amplified and sequenced, and the distribution of sgRNAs before and after the mechanical screen is used to determine the correlation of gene knockouts with mechanical subsets.

Article Snippet: The human CRISPR Brunello lentiviral pooled library was designed to optimize on-target activity and reduce off-target effects (Addgene 73178-LV) .

Techniques: Expressing, Transduction, Selection, FACS, Amplification

Journal: bioRxiv

Article Title: Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins

doi: 10.1101/2025.10.13.682107

Figure Lengend Snippet: ( A ) Schematic representation of selenocysteine decoding. ( B ) GPX4 and GPX1 selenocysteine decoding reporter design. U, selenocysteine; C, cysteine. ( C ) Fluorescence of GPX1, GPX4, and GPX4M HCT116 reporter clones. Mean fluorescence intensity (MFI) was quantified by flow cytometry and normalized to GPX4M. Mean ± SD shown, with individual clones plotted. n=12 for GPX1, n=17 for GPX4, n=10 for GPX4M (biological replicates). ( D ) Sodium selenite dose response of GPX reporters. MFI was normalized to 0 nmol condition. Mean ± SD with four parameter logistic fit plotted. n=3 biological replicates each for GPX1, GPX4, and GPX4M at all tested doses. ( E ) Flow cytometry histograms of reporter cell lines transduced with lentiviral constructs encoding Cas9 and either negative control sgRNA (sgNeg) or sgRNA targeting SECISBP2 . ( F ) Schematic of CRISPR-Cas9 knockout screen. Following transduction of HCT116 reporter clones with a pooled lentiviral CRISPR library, the brightest and dimmest cells were collected by FACS, followed by analysis of sgRNA enrichment in sorted populations by high-throughput sequencing. ( G ) Known factors required for the uptake, biosynthesis and incorporation of selenocysteine. ( H ) and ( I ) Results of the dim screen for factors required for selenocysteine decoding. MAGeCK gene enrichment scores plotted for GPX4 vs. GPX1 reporters (H) or GPX4 vs. GPX4M reporters (I). Known genes in the selenocysteine pathway are labeled in purple.

Article Snippet: A total of 1.4 x 10 8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 μg/ml polybrene at a multiplicity of infection (MOI) between 0.3 to 0.5.

Techniques: Fluorescence, Clone Assay, Flow Cytometry, Transduction, Construct, Negative Control, CRISPR, Knock-Out, Next-Generation Sequencing, Labeling

Journal: bioRxiv

Article Title: Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins

doi: 10.1101/2025.10.13.682107

Figure Lengend Snippet: ( A ) and ( B ) Results of the bright screen for negative regulators of selenocysteine decoding. MAGeCK gene enrichment scores plotted for GPX4 vs. GPX1 reporters (A) or GPX4 vs. GPX4M reporters (B). Significantly enriched genes in either the GPX1 or GPX4 screen (p<0.01) with roles related to mitochondrial function are labeled in blue. ( C ) and ( D ) Flow cytometry histograms of GPX reporter cell lines transduced with lentiviral constructs encoding Cas9 and the indicated sgRNAs. ( E ) Significantly enriched genes in either the GPX1 or GPX4 bright screen (p<0.01) categorized by mitochondrial function. Significant hits in both screens are labeled in bold. ( F ) and ( G ) Significantly enriched genes in either the GPX1 or GPX4 bright screen (p<0.01), plotted as in (A) and (B). Genes with functions related to translation elongation are labeled in red. ( H ) Significantly enriched genes in either the GPX1 or GPX4 bright screen (p<0.01) with functions related to translation elongation. Significant hits in both screens are labeled in bold. ( I ) Flow cytometry histograms of GPX reporter cell lines transduced with lentiviral constructs encoding Cas9 and the indicated sgRNAs.

Article Snippet: A total of 1.4 x 10 8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 μg/ml polybrene at a multiplicity of infection (MOI) between 0.3 to 0.5.

Techniques: Labeling, Flow Cytometry, Transduction, Construct

Journal: bioRxiv

Article Title: Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins

doi: 10.1101/2025.10.13.682107

Figure Lengend Snippet: ( A ) Flow cytometry histograms of GPX reporter cell lines transduced with lentiviral constructs encoding Cas9 and the indicated sgRNAs. ( B ) and ( C ) Western blot analysis (B) and associated quantification (C) of GPX1 and GPX4 protein levels in HCT116 cells following lentiviral delivery of Cas9 and the indicated sgRNAs. Protein abundance was normalized to GAPDH and to sgNeg. Mean ± SD plotted. n=4 biological replicates. P values were calculated by one-sample t-test. *p < 0.05, **p < 0.01. ( D ) Disome profiling (upper) and ZAKα selective ribosome profiling (lower) read coverage on mouse Gpx1 and human GPX1, respectively. For disomes, reads were plotted based on position of the P-site of the leading ribosome. Coding sequences indicated with grey boxes, selenocysteine codons indicated in purple. ( E ) Activation of the ribotoxic stress response. Recruitment of ZAKα to collided ribosomes leads to phosphorylation of p38 and JNK and associated downstream signaling. EDF1 is also recruited to the collided ribosome interface. ( F ) Western blot analysis of p38 phosphorylation in HEK293T cells following treatment for 7 minutes with DMSO, 0.5 µg/ml cycloheximide (CHX), 0.5 µg/ml anisomycin (ANS), 0.75 µM didenmin B (DDB), or 0.75 µM ternatin (TNT). ( G ) Sucrose gradient sedimentation of ribosomes. Cells were treated with DMSO or 0.75 µM TNT for 7 minutes prior to harvesting. ( H ) and ( I ) Western blot analysis of collected sucrose gradient fractions after treatment with DMSO (H) or 0.75 µM ternatin (I). Inp, input prior to sedimentation. ( J ) Ratio of polysome associated EDF1 (fractions 8-12) over cytosolic EDF1 (fraction 1) quantified by western blot. Mean ± SD plotted. n=4 biological replicates. P value was calculated by student’s t-test. **p < 0.01. ( K ) Flow cytometry histograms of GPX reporter cell lines treated with DMSO or 0.5 µM TNT for 48 hours. ( L ) and ( M ) Western blot analysis (L) and associated quantification (M) of GPX1 and GPX4 protein levels in HCT116 cells following treatment with either DMSO or 0.5 µM TNT for 72 hours. Protein abundance was normalized to GAPDH and to DMSO-treated condition. Mean ± SD plotted. n=4 biological replicates. P values were calculated by one-sample t-test. **p < 0.01.

Article Snippet: A total of 1.4 x 10 8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 μg/ml polybrene at a multiplicity of infection (MOI) between 0.3 to 0.5.

Techniques: Flow Cytometry, Transduction, Construct, Western Blot, Quantitative Proteomics, Activation Assay, Phospho-proteomics, Sedimentation

Journal: bioRxiv

Article Title: Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins

doi: 10.1101/2025.10.13.682107

Figure Lengend Snippet: ( A ) Schematic of mitochondrial electron transport chain showing targets of inhibitors and representative genes selected for loss-of-function studies. PCD, piericidin; ROT, rotenone; AMA, antimycin A; OMA, oligomycin A. ( B ) and ( C ) Flow cytometry histograms of GPX reporter cell lines transduced with lentiviral constructs encoding Cas9 and the indicated sgRNAs. ( D ) Flow cytometry histograms of GPX reporter cell lines treated with increasing doses of PCD for 72 hours. ( E ) Total cellular ROS quantification by flow cytometry in HCT116 cells following lentiviral delivery of Cas9 and the indicated sgRNAs. ( F ) Flow cytometry histograms of GPX1 reporter cells transduced with lentiviral constructs encoding Cas9 and the indicated sgRNAs with or without treatment with N-acetyl cysteine (NAC). ( G ) to ( I ) Western blot analysis (G) and associated quantification (H and I) of GPX1 and GPX4 protein levels in HCT116 cells following lentiviral delivery of Cas9 and the indicated sgRNAs. Protein abundance was normalized to GAPDH and to sgNeg. Mean ± SD plotted. n=4 biological replicates. P values were calculated by one-sample t-test. *p < 0.05, **p < 0.01, ***p < 0.001. ( J ) to ( L ) Western blot analysis (J) and associated quantification (K and L) of GPX1 and GPX4 protein levels in HCT116 cells treated with DMSO or 20 nM PCD for 72 hours. Protein abundance was normalized to GAPDH and to mock-treated sample. Mean ± SD plotted. n=6 biological replicates. P values were calculated by one-sample t-test. *p < 0.05, **p < 0.01. ( M ) and ( N ) qRT-PCR analysis of GPX1 (M) and GPX4 (N) transcript abundance in HCT116 cells transduced with lentiviral constructs expressing Cas9 and the indicated sgRNAs. Expression was normalized to housekeeping gene Oaz1 and negative control sgRNA. Mean ± SD plotted. n=4 biological replicates. ( O ) Western blot analysis of SELENOF and SELENOH in HCT116 cells following lentiviral delivery of Cas9 and the indicated sgRNAs. ( P ) and ( Q ) qRT-PCR analysis of SELENOF (M) and SELENOH (N) transcript abundance in HCT116 cells transduced with lentiviral constructs expressing Cas9 and the indicated sgRNAs. Expression was normalized to housekeeping gene Oaz1 and negative control sgRNA. Mean ± SD plotted. n=4 biological replicates.

Article Snippet: A total of 1.4 x 10 8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 μg/ml polybrene at a multiplicity of infection (MOI) between 0.3 to 0.5.

Techniques: Flow Cytometry, Transduction, Construct, Western Blot, Quantitative Proteomics, Quantitative RT-PCR, Expressing, Negative Control

Journal: bioRxiv

Article Title: Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins

doi: 10.1101/2025.10.13.682107

Figure Lengend Snippet: Oxidative stress slows translation elongation in an EEF2K-independent manner. ( A ) and ( B ) Ribosome runoff experiments in HCT116 cells transduced with lentiviral constructs expressing Cas9 and the indicated sgRNAs. Polysome abundance was monitored by sucrose gradient sedimentation before and after 2 minutes of treatment with the translation initiation inhibitor silvestrol (SIL; 2 µM). ( C ) and ( D ) Flow cytometry histograms of GPX reporter cells either untreated (Ctrl) or treated with 10 nM PCD, 0.75 µM TNT, or both PCD and TNT. ( E ) and ( F ) Western blot analysis of EEF2 phosphorylation in HCT116 cells with or without 24 hours of treatment with 20 nM of ETC inhibitors (E) or after treatment with the indicated doses of EEF2K inhibitor A-484954 (EEF2Ki) (F). ( G ) and ( H ) Flow cytometry histograms of GPX1 (G) and GPX4 (H) reporter cell lines treated with vehicle (DMSO) or the indicated compounds. ( I ) to ( K ) Western blot analysis (I) and quantification of GPX1 and GPX4 protein levels (J and K) in EEF2K wild type or knockout HCT116 cells transduced with lentiviral constructs encoding Cas9 and the indicated sgRNAs. Protein abundance was normalized to GAPDH and to sgNeg. Mean ± SD plotted. n=3 biological replicates.

Article Snippet: A total of 1.4 x 10 8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 μg/ml polybrene at a multiplicity of infection (MOI) between 0.3 to 0.5.

Techniques: Transduction, Construct, Expressing, Sedimentation, Flow Cytometry, Western Blot, Phospho-proteomics, Knock-Out, Quantitative Proteomics

Journal: bioRxiv

Article Title: Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins

doi: 10.1101/2025.10.13.682107

Figure Lengend Snippet: ( A ) AF3 model of EEF1G in complex with GSH showing conserved cis-proline at position 59 (red). ( B ) Western blot analysis of glutathione-Sepharose pulldowns from EEF1G WT and EEF1G P59G HCT116 cells. ( C ) Proliferation of parental HCT116 cells (WT) and three independent EEF1G P59G homozygous knock-in clones. ( D ) and ( E ) Ribosome runoff experiments (D) and associated quantification (E) in EEF1G WT or EEF1G P59G HCT116 cells in in the presence or absence of PCD. Polysome abundance was monitored by sucrose gradient sedimentation before and after 2 minutes of treatment with the translation initiation inhibitor silvestrol (SIL; 2 µM). Area under the curve for gradient positions with polysomes (fractions 8-12) was quantified and normalized to 0 min SIL condition. n=3 biological replicates. P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons post-test. n.s., not significant; **p < 0.01. ( F ) to ( H ) Western blot analysis (F) and associated quantification (G and H) of GPX1 and GPX4 protein levels in EEF1G WT or EEF1G P59G HCT116 cells transduced with lentiviral constructs encoding Cas9 and the indicated sgRNAs. Protein abundance was normalized to EEF1G and to sgNeg. Mean ± SD plotted. n=6 biological replicates for EEF1G WT cells and n=8 biological replicates for EEF1G P59G cells from 3 independent knock-in clones. P values were calculated by 2-way ANOVA with Tukey’s multiple comparisons post-test. **p < 0.01, ***p < 0.001.

Article Snippet: A total of 1.4 x 10 8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 μg/ml polybrene at a multiplicity of infection (MOI) between 0.3 to 0.5.

Techniques: Western Blot, Knock-In, Clone Assay, Sedimentation, Transduction, Construct, Quantitative Proteomics