library backbone plasmid lentiguide puro  (Addgene inc)

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

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

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

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

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

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