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lentiviral sgrna library backbone plasmid  (Addgene inc)


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    Addgene inc lentiviral sgrna library backbone plasmid
    (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 <t>sgRNA</t> 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.
    Lentiviral Sgrna Library Backbone Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/library+backbone+plasmid/bio_rxiv__64898__2026__03__06__710083-157-10-15?v=Addgene+inc
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    lentiviral sgrna library backbone plasmid - by Bioz Stars, 2026-06
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    1) Product Images from "Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens"

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

    Journal: bioRxiv

    doi: 10.64898/2026.03.06.710083

    (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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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



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    (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.

    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

    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.

    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

    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.

    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

    (A) Experimental set up of the screen. Cas9-experssing p53 -/- hTERT RPE-1 cells are infected with the TKOv3 lentiviral library. After puromycin selection, infected cells are kept growing for 12 days and then transfected with a gRNA targeting a region of the lentiviral backbone in the proximity of the library sgRNA sequence. Cells are allowed to repair the DSB for 72 hours before genomic DNA purification and amplification of the region containing the library sgRNA sequence and the associated repair outcome. Illumina libraries are then prepared from this amplicon and pair-end sequencing is carried out to link each gene knockout to the DSB repair outcome. (B) Schematic representation of the TKOv3 sgRNA expression cassette. Cut sites used in the screen and location of primers used to generate the Illumina library are indicated. (C) Repair patterns of DSBs generated after transfection of RPE1-Cas9 TP53 -/- TKOv3 cells with each of the three gRNAs used in the screen (see Methods). The relative abundance of each indel is normalized to the total percentage of edited sequences and then classified and ordered by size to facilitate the visualization. Negative indels refer to deletions (red arrow) and positive indels refer to insertions (blue arrow). Data show average of indel frequency, with error bars for ±SD (n= 4 for Cut site 1, n= 7 for Cut site 2, n=5 for Cut site 3). (D, E and F) Top left. Diagram showing the most frequent indels analysed in the screen for cut site 2. Protospacer and PAM sequences are highlighted in blue on the top. The expected cut site for each sgRNA is indicated by a vertical dashed line. Inserted nucleotides are shown in yellow and microhomology regions flanking a deletion are shown in red. The category of each indel is indicated by a coloured square on the left side of the diagram. Top centre. Frequency of each indel, relative to the total frequency of edited events, for the control (blue lines) and the indicated gene sgRNAs (red). Top right. Heatmap showing the Log2 fold change for each indel of the indicated sgRNA relative to the average of control sgRNAs. Bottom. Radar plots for the change of each indel category in the indicated genetic backgrounds. Values show normalized frequency change of indicated sgRNAs relative to controls (blue line). The continuous red line shows average change of the three cut sites combined, while the dashed lines show changes for each independent cut site.

    Journal: bioRxiv

    Article Title: A comprehensive genetic catalog of human double-strand break repair

    doi: 10.1101/2024.08.03.606369

    Figure Lengend Snippet: (A) Experimental set up of the screen. Cas9-experssing p53 -/- hTERT RPE-1 cells are infected with the TKOv3 lentiviral library. After puromycin selection, infected cells are kept growing for 12 days and then transfected with a gRNA targeting a region of the lentiviral backbone in the proximity of the library sgRNA sequence. Cells are allowed to repair the DSB for 72 hours before genomic DNA purification and amplification of the region containing the library sgRNA sequence and the associated repair outcome. Illumina libraries are then prepared from this amplicon and pair-end sequencing is carried out to link each gene knockout to the DSB repair outcome. (B) Schematic representation of the TKOv3 sgRNA expression cassette. Cut sites used in the screen and location of primers used to generate the Illumina library are indicated. (C) Repair patterns of DSBs generated after transfection of RPE1-Cas9 TP53 -/- TKOv3 cells with each of the three gRNAs used in the screen (see Methods). The relative abundance of each indel is normalized to the total percentage of edited sequences and then classified and ordered by size to facilitate the visualization. Negative indels refer to deletions (red arrow) and positive indels refer to insertions (blue arrow). Data show average of indel frequency, with error bars for ±SD (n= 4 for Cut site 1, n= 7 for Cut site 2, n=5 for Cut site 3). (D, E and F) Top left. Diagram showing the most frequent indels analysed in the screen for cut site 2. Protospacer and PAM sequences are highlighted in blue on the top. The expected cut site for each sgRNA is indicated by a vertical dashed line. Inserted nucleotides are shown in yellow and microhomology regions flanking a deletion are shown in red. The category of each indel is indicated by a coloured square on the left side of the diagram. Top centre. Frequency of each indel, relative to the total frequency of edited events, for the control (blue lines) and the indicated gene sgRNAs (red). Top right. Heatmap showing the Log2 fold change for each indel of the indicated sgRNA relative to the average of control sgRNAs. Bottom. Radar plots for the change of each indel category in the indicated genetic backgrounds. Values show normalized frequency change of indicated sgRNAs relative to controls (blue line). The continuous red line shows average change of the three cut sites combined, while the dashed lines show changes for each independent cut site.

    Article Snippet: RPE1 Cas9 TKOv3-NT and RPE1 Cas9 TP53 -/- TKOv3-NT cell lines were generated by lentiviral transduction using the TKOv3 library backbone plasmid (pLCKO2, Addgene #125518) with a non-targeting gRNA sequence cloned into the BsmB1 restriction sites.

    Techniques: Infection, Selection, Transfection, Sequencing, DNA Purification, Amplification, Gene Knockout, Expressing, Generated, Control

    (A) Summary of results obtained in the screen for XLF and PAXX, as in ( , E and F). Only TKOv3 Cut 2 is shown. (B) Impact of XLF, PAXX and POLL absence on the repair profile at TKOv3 Cut site 2 in the indicated RPE1- Cas9 TKOv3-NT clones. Experiments are carried out as in . Data show average values normalized to total editing efficiency. n = 6, error bars show ±SD. (C) Model for PAXX/XLF opposing effect on the equilibrium between open and closed conformations of NHEJ synapses. Particle distribution information from published cryo-EM studies ( , , – ) was used to evaluate the effect of PAXX and XLF on the equilibrium and is shown here as a percentage of the total long-range particles found in those datasets (see Methods for a description of data analysis and selection). Representative structures for the long-range open (8BH3) and closed (8BHV) conformations, short-range complex (7LSY), MRE11 (1XSP) and POLL (8BAH) are shown to illustrate the model.

    Journal: bioRxiv

    Article Title: A comprehensive genetic catalog of human double-strand break repair

    doi: 10.1101/2024.08.03.606369

    Figure Lengend Snippet: (A) Summary of results obtained in the screen for XLF and PAXX, as in ( , E and F). Only TKOv3 Cut 2 is shown. (B) Impact of XLF, PAXX and POLL absence on the repair profile at TKOv3 Cut site 2 in the indicated RPE1- Cas9 TKOv3-NT clones. Experiments are carried out as in . Data show average values normalized to total editing efficiency. n = 6, error bars show ±SD. (C) Model for PAXX/XLF opposing effect on the equilibrium between open and closed conformations of NHEJ synapses. Particle distribution information from published cryo-EM studies ( , , – ) was used to evaluate the effect of PAXX and XLF on the equilibrium and is shown here as a percentage of the total long-range particles found in those datasets (see Methods for a description of data analysis and selection). Representative structures for the long-range open (8BH3) and closed (8BHV) conformations, short-range complex (7LSY), MRE11 (1XSP) and POLL (8BAH) are shown to illustrate the model.

    Article Snippet: RPE1 Cas9 TKOv3-NT and RPE1 Cas9 TP53 -/- TKOv3-NT cell lines were generated by lentiviral transduction using the TKOv3 library backbone plasmid (pLCKO2, Addgene #125518) with a non-targeting gRNA sequence cloned into the BsmB1 restriction sites.

    Techniques: Clone Assay, Cryo-EM Sample Prep, Selection

    (A) Effect of GET3 on overall editing efficiency at TKOv3 Cut 2 in RPE1- Cas9 TKOv3-NT cells. Experiments carried out as in . Two GET3 -/- clones are tested. n = 12 for WT and 4 for GET3 -/- clones. Error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (B) Cas9 cutting efficiency at TKOv3 Cut site 2 estimated by qPCR. Primers annealing at both sides of the cut site are used to determine the percentage of cut molecules 6h after gRNA transfection in the indicated cell lines. Amplification levels were normalized to the endogenous gene GREB1. n = 6, error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (C) 53BP1 foci number per cell 12h after transfection with a multi-target gRNA (1.5 nM) in the indicated cell lines. Data shows foci count of two independent replicates merged. Statistical analysis was performed using One-way ANOVA. (D) Heatmap showing indel frequency variation (Log2 fold change) in the three cut sites of the screen for the reduced editing efficiency gene cluster . Two insertion events in the TKOv3 Cut site 2 that are consistently reduced or increased across most knockouts in this group are marked with a blue and a red triangle, respectively. (E) Frequency of +2 (left) and +1 (right) nucleotide insertions, relative to the overall editing efficiency, after transfection with the TKOv3 Cut site 2 gRNA in RPE1- Cas9 TKOv3 GET 3 -/- clones compared to wild type cells. Experiments carried out as in . n = 12 for WT and 4 for GET3 -/- clones. Error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (F) Variation in the frequency of +1 and +2 nucleotide insertions produced after transfection with decreasing concentrations (2-fold serial dilutions from our standard gRNA final concentration, 12 nM) of gRNA targeting TKOv3 Cut site 2 in RPE1- Cas9 TKOv3-NT Cells. n = 3, error bars show ±SD. (G) In-vitro Cas9 DNA cleavage assay showing differential gRNA mismatch tolerance. Schematic representation of the two constructs containing Cut site 1 and 2 sequence either unedited or including the most common +1 nucleotide insertion outcome observed in the screen data (left) (see Methods). Agarose gel showing digestion of the constructs with increasing concentrations of Cas9 ribonucleoparticle bearing gRNA targeting either Cut site 1 or 2 (right).

    Journal: bioRxiv

    Article Title: A comprehensive genetic catalog of human double-strand break repair

    doi: 10.1101/2024.08.03.606369

    Figure Lengend Snippet: (A) Effect of GET3 on overall editing efficiency at TKOv3 Cut 2 in RPE1- Cas9 TKOv3-NT cells. Experiments carried out as in . Two GET3 -/- clones are tested. n = 12 for WT and 4 for GET3 -/- clones. Error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (B) Cas9 cutting efficiency at TKOv3 Cut site 2 estimated by qPCR. Primers annealing at both sides of the cut site are used to determine the percentage of cut molecules 6h after gRNA transfection in the indicated cell lines. Amplification levels were normalized to the endogenous gene GREB1. n = 6, error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (C) 53BP1 foci number per cell 12h after transfection with a multi-target gRNA (1.5 nM) in the indicated cell lines. Data shows foci count of two independent replicates merged. Statistical analysis was performed using One-way ANOVA. (D) Heatmap showing indel frequency variation (Log2 fold change) in the three cut sites of the screen for the reduced editing efficiency gene cluster . Two insertion events in the TKOv3 Cut site 2 that are consistently reduced or increased across most knockouts in this group are marked with a blue and a red triangle, respectively. (E) Frequency of +2 (left) and +1 (right) nucleotide insertions, relative to the overall editing efficiency, after transfection with the TKOv3 Cut site 2 gRNA in RPE1- Cas9 TKOv3 GET 3 -/- clones compared to wild type cells. Experiments carried out as in . n = 12 for WT and 4 for GET3 -/- clones. Error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (F) Variation in the frequency of +1 and +2 nucleotide insertions produced after transfection with decreasing concentrations (2-fold serial dilutions from our standard gRNA final concentration, 12 nM) of gRNA targeting TKOv3 Cut site 2 in RPE1- Cas9 TKOv3-NT Cells. n = 3, error bars show ±SD. (G) In-vitro Cas9 DNA cleavage assay showing differential gRNA mismatch tolerance. Schematic representation of the two constructs containing Cut site 1 and 2 sequence either unedited or including the most common +1 nucleotide insertion outcome observed in the screen data (left) (see Methods). Agarose gel showing digestion of the constructs with increasing concentrations of Cas9 ribonucleoparticle bearing gRNA targeting either Cut site 1 or 2 (right).

    Article Snippet: RPE1 Cas9 TKOv3-NT and RPE1 Cas9 TP53 -/- TKOv3-NT cell lines were generated by lentiviral transduction using the TKOv3 library backbone plasmid (pLCKO2, Addgene #125518) with a non-targeting gRNA sequence cloned into the BsmB1 restriction sites.

    Techniques: Clone Assay, Transfection, Amplification, Produced, Concentration Assay, In Vitro, DNA Cleavage Assay, Construct, Sequencing, Agarose Gel Electrophoresis

    (A) Summary of results obtained in the screen for HLTF, as in . Only data for TKOv3 Cut site 2 is displayed. (B) Impact of knocking HLTF out on the repair profile produced at TKOv3 Cut site 2 in RPE1- Cas9 TKOv3 cells. Assay performed as in . Three independent HLTF -/- clones are shown. n = 3, except for clone 9 (n = 4), error bars show ±SD. (C) Effect of PCNA on the repair profile of wild type and HLTF -/- RPE1- Cas9 TKOv3 cells. After transfection with NT or PCNA -targeting gRNAs, cells are arrested in G0/G1 by confluency for 12 days and then transfected with the TKOv3 Cut site 2 gRNA. Genomic DNA is purified three days later. n = 3 for WT + NT gRNA, n = 5 for WT + PCNA gRNA, n = 2 for HLTF -/- + NT gRNA and n = 5 for HLTF -/- + PCNA gRNA. Error bars show ±SD. (D) Repair pattern obtained at AAVS1 cut site 4 in the RPE1- Cas9 HLTF -/- cell line complemented with the indicated versions of HLTF . Doxycycline is added to induce HLTF expression (figure S5C) 24 hours before transfection with AAVS1 cut site 4 gRNA, and it is maintained until genomic DNA purification (3 days after transfection). Only most frequent indels are shown. n = 3 except for the RING mutant (n = 2). Error bars show ±SD. (E) ChIP q-PCR experiments showing HLTF binding to TKOv3 Cut site 2 in the indicated genotypes at 0 and 3 hours after transfection with TKOv3 Cut site 2 gRNA. GREB1 locus was used as a control site. n = 3, error bars show ±SD. Statistical analysis was performed using Two-way ANOVA. (F) (left) ChIP q-PCR experiments showing Cas9 binding to TKOv3 Cut site 2 in the indicated genotypes at different time points after transfection with the TKOv3 Cut site 2 gRNA. GREB1 locus was used as a control site. (right) Cas9 cleavage efficiency at TKOv3 Cut site 2 estimated by qPCR (as in ) in the same samples used for Cas9 ChIP experiments in wild type and HLTF -/- backgrounds. n = 3, error bars show ±SD. Statistical analysis was performed using Two-way ANOVA. (G) Experimental layout diagram for the in vitro removal of post-cleavage Cas9 RNP by HLTF (left, see figure S5F and Methods). Immobilized dsDNA, containing the TKOv3 Cut 2 gRNA target sequence, is digested by the addition of TKOv3 Cut 2 gRNA-Cas9 RNP. Retained Cas9 RNP is then released by incorporation of increasing bacteria-purified HLTF concentrations to the mix. (Right) Representative electrophoresis gel image of flow-through material after incubation with HLTF. Normalized quantification of the released Cas9 bands is shown below the electrophoresis image.

    Journal: bioRxiv

    Article Title: A comprehensive genetic catalog of human double-strand break repair

    doi: 10.1101/2024.08.03.606369

    Figure Lengend Snippet: (A) Summary of results obtained in the screen for HLTF, as in . Only data for TKOv3 Cut site 2 is displayed. (B) Impact of knocking HLTF out on the repair profile produced at TKOv3 Cut site 2 in RPE1- Cas9 TKOv3 cells. Assay performed as in . Three independent HLTF -/- clones are shown. n = 3, except for clone 9 (n = 4), error bars show ±SD. (C) Effect of PCNA on the repair profile of wild type and HLTF -/- RPE1- Cas9 TKOv3 cells. After transfection with NT or PCNA -targeting gRNAs, cells are arrested in G0/G1 by confluency for 12 days and then transfected with the TKOv3 Cut site 2 gRNA. Genomic DNA is purified three days later. n = 3 for WT + NT gRNA, n = 5 for WT + PCNA gRNA, n = 2 for HLTF -/- + NT gRNA and n = 5 for HLTF -/- + PCNA gRNA. Error bars show ±SD. (D) Repair pattern obtained at AAVS1 cut site 4 in the RPE1- Cas9 HLTF -/- cell line complemented with the indicated versions of HLTF . Doxycycline is added to induce HLTF expression (figure S5C) 24 hours before transfection with AAVS1 cut site 4 gRNA, and it is maintained until genomic DNA purification (3 days after transfection). Only most frequent indels are shown. n = 3 except for the RING mutant (n = 2). Error bars show ±SD. (E) ChIP q-PCR experiments showing HLTF binding to TKOv3 Cut site 2 in the indicated genotypes at 0 and 3 hours after transfection with TKOv3 Cut site 2 gRNA. GREB1 locus was used as a control site. n = 3, error bars show ±SD. Statistical analysis was performed using Two-way ANOVA. (F) (left) ChIP q-PCR experiments showing Cas9 binding to TKOv3 Cut site 2 in the indicated genotypes at different time points after transfection with the TKOv3 Cut site 2 gRNA. GREB1 locus was used as a control site. (right) Cas9 cleavage efficiency at TKOv3 Cut site 2 estimated by qPCR (as in ) in the same samples used for Cas9 ChIP experiments in wild type and HLTF -/- backgrounds. n = 3, error bars show ±SD. Statistical analysis was performed using Two-way ANOVA. (G) Experimental layout diagram for the in vitro removal of post-cleavage Cas9 RNP by HLTF (left, see figure S5F and Methods). Immobilized dsDNA, containing the TKOv3 Cut 2 gRNA target sequence, is digested by the addition of TKOv3 Cut 2 gRNA-Cas9 RNP. Retained Cas9 RNP is then released by incorporation of increasing bacteria-purified HLTF concentrations to the mix. (Right) Representative electrophoresis gel image of flow-through material after incubation with HLTF. Normalized quantification of the released Cas9 bands is shown below the electrophoresis image.

    Article Snippet: RPE1 Cas9 TKOv3-NT and RPE1 Cas9 TP53 -/- TKOv3-NT cell lines were generated by lentiviral transduction using the TKOv3 library backbone plasmid (pLCKO2, Addgene #125518) with a non-targeting gRNA sequence cloned into the BsmB1 restriction sites.

    Techniques: Produced, Clone Assay, Transfection, Purification, Expressing, DNA Purification, Mutagenesis, Binding Assay, Control, In Vitro, Sequencing, Bacteria, Electrophoresis, Incubation

    (A) Impact of knocking POLQ out on the repair profile observed at TKOv3 Cut site 2 in RPE1- Cas9 TKOv3 cells. Assay performed as in . Two independent POLQ -/- clones are shown. n = 11 for wild type and 6 for POLQ -/- clones. Error bars show ±SD. (B) STRING analysis of interactions between selected proteins (i. e., POLQ PCC > 0.45). Only functionally connected genes are annotated. (C) Summary of screen results for cut site 2 for BLM , TADA1 and FANCF . Displayed as in .

    Journal: bioRxiv

    Article Title: A comprehensive genetic catalog of human double-strand break repair

    doi: 10.1101/2024.08.03.606369

    Figure Lengend Snippet: (A) Impact of knocking POLQ out on the repair profile observed at TKOv3 Cut site 2 in RPE1- Cas9 TKOv3 cells. Assay performed as in . Two independent POLQ -/- clones are shown. n = 11 for wild type and 6 for POLQ -/- clones. Error bars show ±SD. (B) STRING analysis of interactions between selected proteins (i. e., POLQ PCC > 0.45). Only functionally connected genes are annotated. (C) Summary of screen results for cut site 2 for BLM , TADA1 and FANCF . Displayed as in .

    Article Snippet: RPE1 Cas9 TKOv3-NT and RPE1 Cas9 TP53 -/- TKOv3-NT cell lines were generated by lentiviral transduction using the TKOv3 library backbone plasmid (pLCKO2, Addgene #125518) with a non-targeting gRNA sequence cloned into the BsmB1 restriction sites.

    Techniques: Clone Assay

    (A) Summary of screening results for VHL and EGLN1 , as in . Only TKOv3 Cut 2 data is shown. (B) Effect of indicated de-novo designed gRNAs transfection on the repair profile produced at TKOv3 Cut site 2 in RPE1- Cas9 TKOv3-NT cells. Assay carried out as in figure S3A (see also Methods). n = 3, error bars show ±SD. (C) Effect of the indicated gRNAs transfection on the percentage of insertions relative to all indels obtained at the TKOv3 Cut site 2 and at different endogenous AAVS1 Cut sites. Assay carried out as in figure S3A. n = 3, error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (D) Effect of the indicated gRNAs co-transfection on the percentage of insertions relative to all indels obtained at the AAVS1 Cut site 4. Assay carried out as in figure S3A. n = 3, error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (E) Comparison between the relative frequency (i.e., prevalence) of tumour samples with detectable ID11 signature activity in 144 ccRCC samples and 2,464 tumour samples from other 36 tumour types represented in PCAWG. P-value is calculated using Fisher’s exact test. (F) Comparison between ID11 signature activity in 144 ccRCC samples and 2,464 tumour samples from other 36 tumour types in PCAWG. P-value is calculated using unpaired, two-sided Wilcoxon test. (G-H) Comparison of VHL expression (FPKM counts) (G) and hypoxia scores (H), as previously estimated , using a reported signature , with activity of ID11 in 1,067 non-ccRCC tumour samples with available RNA-Seq expression in PCAWG. VHL expression and hypoxia scores in active ID11 (n = 37, ID11 signature activity > 0) and inactive ID11 (n = 1030, ID11 signature activity = 0) are compared using an unpaired, two-sided Wilcoxon test.

    Journal: bioRxiv

    Article Title: A comprehensive genetic catalog of human double-strand break repair

    doi: 10.1101/2024.08.03.606369

    Figure Lengend Snippet: (A) Summary of screening results for VHL and EGLN1 , as in . Only TKOv3 Cut 2 data is shown. (B) Effect of indicated de-novo designed gRNAs transfection on the repair profile produced at TKOv3 Cut site 2 in RPE1- Cas9 TKOv3-NT cells. Assay carried out as in figure S3A (see also Methods). n = 3, error bars show ±SD. (C) Effect of the indicated gRNAs transfection on the percentage of insertions relative to all indels obtained at the TKOv3 Cut site 2 and at different endogenous AAVS1 Cut sites. Assay carried out as in figure S3A. n = 3, error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (D) Effect of the indicated gRNAs co-transfection on the percentage of insertions relative to all indels obtained at the AAVS1 Cut site 4. Assay carried out as in figure S3A. n = 3, error bars show ±SD. Statistical analysis was performed using One-way ANOVA. (E) Comparison between the relative frequency (i.e., prevalence) of tumour samples with detectable ID11 signature activity in 144 ccRCC samples and 2,464 tumour samples from other 36 tumour types represented in PCAWG. P-value is calculated using Fisher’s exact test. (F) Comparison between ID11 signature activity in 144 ccRCC samples and 2,464 tumour samples from other 36 tumour types in PCAWG. P-value is calculated using unpaired, two-sided Wilcoxon test. (G-H) Comparison of VHL expression (FPKM counts) (G) and hypoxia scores (H), as previously estimated , using a reported signature , with activity of ID11 in 1,067 non-ccRCC tumour samples with available RNA-Seq expression in PCAWG. VHL expression and hypoxia scores in active ID11 (n = 37, ID11 signature activity > 0) and inactive ID11 (n = 1030, ID11 signature activity = 0) are compared using an unpaired, two-sided Wilcoxon test.

    Article Snippet: RPE1 Cas9 TKOv3-NT and RPE1 Cas9 TP53 -/- TKOv3-NT cell lines were generated by lentiviral transduction using the TKOv3 library backbone plasmid (pLCKO2, Addgene #125518) with a non-targeting gRNA sequence cloned into the BsmB1 restriction sites.

    Techniques: Transfection, Produced, Cotransfection, Comparison, Activity Assay, Expressing, RNA Sequencing