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: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

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: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

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: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

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: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

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

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: EXO1 and CHAF1A are recruited to R-loops and synergistically suppress R-loop accumulation. ( A ) BrdU alkaline comet assay showing that CHAF1A depletion causes increased accumulation of replication-associated single stranded DNA lesions in both EXO1-knockout lines compared to WT HeLa cells. At least 100 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. ( B, C ) S9.6 PLA experiments showing increased EXO1 ( B ) and CHAF1A ( C ) recruitment to R-loops in HeLa cells. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( D, E ) S9.6-dsDNA PLA experiments showing increased R-loops in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

Techniques: Alkaline Single Cell Gel Electrophoresis, Knock-Out, MANN-WHITNEY, Two Tailed Test, Over Expression

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: Concomitant depletion of EXO1 and CHAF1A causes the accumulation of R-loop-associated DNA damage. ( A ) S9.6-γH2AX PLA experiments showing increased R-loop-associated DNA damage in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). The siCHAF1A/siControl mean ratios are also presented. ( B–D ) γH2AX immunofluorescence showing that CHAF1A depletion causes increased DNA damage in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( B, D ) and representative micrographs with scale bars representing 10 µm ( C ) are shown. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( E–G ) Neutral comet assays showing that CHAF1A depletion causes increased DSB formation in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( E, G ) and representative micrographs with scale bars representing 10 µm ( F ) are shown. At least 100 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, two-tailed).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

Techniques: Over Expression, Two Tailed Test, Immunofluorescence, Knock-Out, MANN-WHITNEY

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: To knock-out EXO1 in HeLa cells, a commercially available CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology sc-402356) was used, as we previously described [ ].

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: To knock-out EXO1 in HeLa cells, a commercially available CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology sc-402356) was used, as we previously described [ ].

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: To knock-out EXO1 in HeLa cells, a commercially available CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology sc-402356) was used, as we previously described [ ].

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: To knock-out EXO1 in HeLa cells, a commercially available CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology sc-402356) was used, as we previously described [ ].

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

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: EXO1 and CHAF1A are recruited to R-loops and synergistically suppress R-loop accumulation. ( A ) BrdU alkaline comet assay showing that CHAF1A depletion causes increased accumulation of replication-associated single stranded DNA lesions in both EXO1-knockout lines compared to WT HeLa cells. At least 100 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. ( B, C ) S9.6 PLA experiments showing increased EXO1 ( B ) and CHAF1A ( C ) recruitment to R-loops in HeLa cells. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( D, E ) S9.6-dsDNA PLA experiments showing increased R-loops in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired).

Article Snippet: To knock-out EXO1 in HeLa cells, a commercially available CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology sc-402356) was used, as we previously described [ ].

Techniques: Alkaline Single Cell Gel Electrophoresis, Knock-Out, MANN-WHITNEY, Two Tailed Test, Over Expression

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: Concomitant depletion of EXO1 and CHAF1A causes the accumulation of R-loop-associated DNA damage. ( A ) S9.6-γH2AX PLA experiments showing increased R-loop-associated DNA damage in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). The siCHAF1A/siControl mean ratios are also presented. ( B–D ) γH2AX immunofluorescence showing that CHAF1A depletion causes increased DNA damage in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( B, D ) and representative micrographs with scale bars representing 10 µm ( C ) are shown. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( E–G ) Neutral comet assays showing that CHAF1A depletion causes increased DSB formation in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( E, G ) and representative micrographs with scale bars representing 10 µm ( F ) are shown. At least 100 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, two-tailed).

Article Snippet: To knock-out EXO1 in HeLa cells, a commercially available CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology sc-402356) was used, as we previously described [ ].

Techniques: Over Expression, Two Tailed Test, Immunofluorescence, Knock-Out, MANN-WHITNEY