brunello lentiviral library  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Indel pattern-guided repair mapping reveals genome-wide DNA repair networks in CRISPR/Cas9 editing

doi: 10.1093/nar/gkag260

Figure Lengend Snippet: IPGRM as a genome-wide platform for systematic dissection of DSB repair mechanisms. ( A ) Schematic of vectors encoding the GeCKOv2 CRISPR knockout library with secondary Cas9 cleavage sites (sg3 or sg81) positioned adjacent to library-encoded sgRNAs. ( B ) Composition of the GeCKOv2 library, including coding genes, pre-miRNA loci, and non-targeting controls. ( C ) Workflow of IPGRM. HEK293T cells were transduced with the GeCKOv2 library and subsequently edited at sg3 or sg81 loci. Repair outcomes were captured by PCR amplicon sequencing and classified into seven indel patterns, allowing each sgRNA-mediated knockout to be directly linked to a corresponding repair outcome profile. Insertion length distributions in the sg3 ( D ) and sg81 ( E ) libraries. Deletion length distributions in the sg3 ( F ) and sg81 ( G ) libraries. Sequence profiles of the most frequent deletions: 3 bp in the sg3 library ( H ) and 5 bp in the sg81 library ( I ). Microhomologous bases are indicated by underlining. ( J ) Proportional distributions of the seven indel patterns in the sg3 and sg81 libraries.

Article Snippet: The human GeCKOv2 lentiviral library (GENEWIZ, Suzhou), containing 122 756 sgRNAs targeting 19 050 genes (6 sgRNAs per gene), 1864 miRNAs (4 sgRNAs per miRNA), and 1000 NonTarget controls, was transduced into 3 × 108 HEK293T cells at an MOI of 0.3.

Techniques: Genome Wide, Dissection, CRISPR, Knock-Out, Transduction, Amplification, Sequencing

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: PARG inhibition sequesters nuclear PAR-binding proteins, including XRCC1 and its partners, into nuclear condensates to elicit cytotoxicity

doi: 10.64898/2026.03.18.712393

Figure Lengend Snippet: A. V-abl B cells expressing Flag-tagged Cas9 were infected with a BFP-expressing lentiviral gRNA library. BFP+ cells were sorted and treated with doxycycline for five days to induce Cas9 expression. Indicated inhibitors treatment was performed at IC90 for six days. Then, DNA was collected from the cells for sequencing and analysis using the MAGeCK pipeline. B. Scatter plot of total CRISPR genes Z-scores for Niraparib and Olaparib IC90 treatments. Simple linear regression and Pearson correlation test were performed to determine R. C. Scatter plot of total CRISPR genes Z-scores for PDD0017273 and Olaparib IC90 treatments. Simple linear regression and Pearson correlation test were performed to determine R. D. Summary table of Pearson R coefficient for linear regression between cell lines sensitivity to PDD, olaparib, nirabarib and talazoparib. Depmap sensitivity score (PRISM repurposing data) of 536 cell lines to the indicated compound. Simple linear regression and Pearson correlation test were performed to determine R. Colored scaled is represented on the right and R coefficients are written in white. E. Heatmap representing z-score (color range) and False Discovery Rate FDR (size) for the genes targeting indicated in rows, in cells challenged with inhibitor indicated above, in columns. Gene implicated in homologous recombination are displayed. F. CRISPR screen z-scores ranking of co-essential genes with PDD00017273 treatment. SSBR related genes are shown in red, ADP-ribosylation related genes are shown in dark blue, HR related genes are shown in light grey.

Article Snippet: 100 million cells were infected with a BFP-expressing gRNA lentiviral library (Addgene Pooled Library #67988).

Techniques: Expressing, Infection, Sequencing, CRISPR, Homologous Recombination

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: (A) Summary of gene deletion vs. gene overexpression approaches. (B) Rationale for the present study. (C) Experimental outline of the present study. (D) HEK293 and Jurkat cells were inoculated with different volumes of Ebola or rabies pseudovirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This revealed that Jurkat cells are largely refractory to Ebola or rabies pseudovirus entry, relative to HEK293 cells. (E) Construction of a clonal Jurkat cell line, known as “Jurkat C6”, stably expressing a degron-tagged CRISPRa construct ( left ). Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence or absence of TMP (1 μM) for 3 days, followed by flow cytometry to detect hCD19 expression ( right ). (F) At each of the indicated points of the genome-wide CRISPRa screen, the cell population was challenged with Ebola or rabies pseudovirus, and cell infectivity was evaluated by flow cytometry. Upon successive rounds of the screen, the cell population became progressively more susceptible to infection to either Ebola or rabies pseudovirus, respectively. (G) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for Ebola pseudovirus entry. (H) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for rabies pseudovirus entry.

Article Snippet: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

Techniques: Over Expression, Marker, Mutagenesis, Flow Cytometry, Infection, Stable Transfection, Expressing, Construct, Transduction, Genome Wide

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) Plasmids used to pseudotype non-replicating lentiviruses with either Ebola or rabies envelope proteins. EBOV-GP: glycoprotein of Ebola virus, Makona variant. RABV-GP N2C: glycoprotein of rabies virus, N2C variant. B) HEK293 and Jurkat cells were inoculated with different volumes of VSV envelope protein-pseudotyped lentivirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This served as a positive control to confirm that Jurkat cells and HEK293 cells are both susceptible to VSV pseudovirus entry. C) Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence of different TMP concentrations (0-4 μM) for 2-3 days, followed by flow cytometry to detect human CD19 expression.

Article Snippet: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

Techniques: Virus, Variant Assay, Marker, Mutagenesis, Flow Cytometry, Infection, Positive Control, Transduction, Expressing

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) NGFR was expressed in Jurkat C6 cells using CRISPRa, or alternatively, Jurkat cells using cDNA expression. NGFR -expressing or control cells were then inoculated with rabies pseudovirus encoding mCD19t. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that NGFR expression significantly increased the susceptibility of Jurkat cells to rabies pseudovirus infection. As positive controls, flow cytometry was used to confirm successful delivery of the sgRNA construct as part of the CRISPRa workflow (as denoted by BFP expression) and that NGFR was expressed (upon cDNA expression). B) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression. L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with rabies pseudovirus encoding hEGFRt (a truncated mutant of human EGFR). Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of Jurkat cells and primary T cells to Ebola pseudovirus infection. Cells expressing the highest levels of L-SIGN and DC-SIGN were preferentially infected by Ebola pseudovirus. C) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression, and then L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with GFP -expressing Ebola virus or zsGreen -expressing Sudan virus under BSL4 containment. On days 0, 1, and 2 post-infection, flow cytometry was performed to determine the percentage of infected cells and qPCR was performed on cell culture supernatants to quantify viral genome replication. This revealed that L-SIGN or DC-SIGN expression enabled authentic Ebola and Sudan virus entry into primary human T cells, but viral genome replication was impaired, perhaps reflective of cell-intrinsic restriction factors.

Article Snippet: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

Techniques: Expressing, Control, Flow Cytometry, Infection, Construct, Mutagenesis, Virus, Cell Culture

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