Journal: Journal of Virology

Article Title: The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle

doi: 10.1128/jvi.00180-23

Figure Lengend Snippet: Role of ERES and ERGIC in HCV entry and replication. (A) Bar graph depicting the relative fold change in luciferase reporter activity in SEC16A, ERGIC-53, TFG, and Claudin-1 (CLDN-1) knockdown cells transduced with HCV pseudotyped particles (HCVpp). (B) Western blot analysis depicting the expression status of cell surface receptors in Huh-7 cells knockdown for SEC16A, ERGIC-53, TFG, and Claudin-1. GAPDH was used as an internal loading control. (C) Bar graphs representing the relative fold change in luciferase reporter activity in HCV genotype 2a subgenomic replicon (SGR-Feo) cells transfected with siNT, siSEC16A, siERGIC-53, and siTFG at 48 h posttransfection. (D) Western blot analysis depicting the knockdown efficiency from the subgenomic replicon cells transfected with respective siRNA. (E) Illustration representing the experimental timeline and approach used to determine the role of ERES, ERGIC, and TFG in HCV replication in the HCV cell culture model. (F) Bar graphs representing the fold change relative to siNT in HCV genome copies at 18 hpi. (G) Immunofluorescence images and Western blot analysis depicting the overexpression of SEC16A-GFP or EV-GFP and the SEC16A knockdown efficiency. (H) Bar graph depicting the relative fold change in luciferase reporter activity in SGR-Feo cells transfected with either siNT, siNT plus empty vector (EV-GFP), siSEC16A, or siSEC16A plus SEC16A-GFP. The respective overexpression vectors were transfected at 24 h posttransfection with the respective siRNAs. The luciferase reporter activity was determined 72 h after siRNA transfection. Scale bar, 100 μm. The data presented are means ± the SEM of three independent experiments. Statistical analysis was done using a Student t test (ns, nonsignificant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001).

Article Snippet: Lentivirus particles harboring the respective shRNA were produced in 293FT cells by cotransfection of lentiviral shRNA vector (pLKO.1 puro), pCMVR8.74 (Addgene 22036), and pMD2.G (Addgene 12259) with a Cal-Phos mammalian transfection kit according to the manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Knockdown, Transduction, Western Blot, Expressing, Control, Transfection, Cell Culture, Immunofluorescence, Over Expression, Plasmid Preparation

Journal: Journal of Virology

Article Title: The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle

doi: 10.1128/jvi.00180-23

Figure Lengend Snippet: ERES and ERGIC role in HCV assembly and secretion. (A) Illustration representing the experimental timeline and approach used to determine the role of ERES, ERGIC, and TFG in HCV assembly and secretion. (B) Western blot analysis depicting the knockdown efficiency of SEC16A, ERGIC-53, and TFG in cells transfected with respective siRNA. β-Actin and GAPDH were used as internal loading controls. (C to F) Bar graphs representing the relative fold change in the intracellular (C) and extracellular (E) HCV genome copies and the relative change in intracellular (D) and extracellular (F) infectious viral titers in HCV-infected cells at 96 hpi and 48 h posttransfection with siRNAs against SEC16A, ERGIC-53, and TFG. (G and H) Bar graphs representing the relative fold change in the level of intra- and extracellular viral RNA levels (G) and infectious virus titers (H) in HCV-infected cells 48 hpi subjected to 24-h treatment with the indicated concentrations of FLI-06. Dimethyl sulfoxide was used as vehicle control (VC). (I) Confocal images representing the ERGIC (green) morphology and HCV envelope (E2, red) distribution in cells treated with DMSO (VC) or 10 μM FLI-06 for 24 h. Scale bar, 10 μm. The data presented are means ± the SEM of three independent experiments. Statistical analysis was done by using a Student t test (ns, nonsignificant; ****, P < 0.0001).

Article Snippet: Lentivirus particles harboring the respective shRNA were produced in 293FT cells by cotransfection of lentiviral shRNA vector (pLKO.1 puro), pCMVR8.74 (Addgene 22036), and pMD2.G (Addgene 12259) with a Cal-Phos mammalian transfection kit according to the manufacturer’s instructions.

Techniques: Western Blot, Knockdown, Transfection, Infection, Virus, Control

Journal: Journal of Virology

Article Title: The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle

doi: 10.1128/jvi.00180-23

Figure Lengend Snippet: SEC16A and TFG overexpression rescue the HCV assembly and release in SEC16A and TFG knockdown cells. (A to D) HCV-infected Huh7 cells at 48 hpi were transfected with siNT, siSEC16A, siTFG siRNAs and 24 h later transfected with the SEC16A-GFP (A and B) or TFG-FLAG (C and D), respectively. siNT-transfected cells were transfected with corresponding empty vectors (EV-GFP or EV) at 24 h posttransfection with the non-targeting siRNA. HCV infectious titers were determined in the culture supernatants, and cells by using an FFU assay. The bar graphs depict the intra- and extracellular viral titers from the HCV-infected Huh7 cells subjected to respective experimental conditions. (E) Western blot analysis and immunofluorescence images depicting the SEC16A knockdown efficiency and overexpression of SEC16A-GFP or EV-GFP. NS5A and GAPDH were used as an HCV infection marker and an internal loading control, respectively. Scale bar, 100 μm. (F) Western blot analysis depicting the TFG knockdown efficiency and overexpression of TFG-FLAG in HCV-infected Huh7 cells. NS3 and GAPDH were used as an HCV infection marker and an internal loading control, respectively.

Article Snippet: Lentivirus particles harboring the respective shRNA were produced in 293FT cells by cotransfection of lentiviral shRNA vector (pLKO.1 puro), pCMVR8.74 (Addgene 22036), and pMD2.G (Addgene 12259) with a Cal-Phos mammalian transfection kit according to the manufacturer’s instructions.

Techniques: Over Expression, Knockdown, Infection, Transfection, Western Blot, Immunofluorescence, Marker, Control

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Effect of SETDB1 depletion on H3K9me3 profiles in untreated and LPS-stimulated macrophages. ( A ) Snapshot showing H3K9me3 profiles in control and SETDB1-depleted untreated mouse bone marrow-derived macrophages in a 7-Mb genomic region. Compartments A (red) and B (blue) are indicated. Normalized H3K9me3 counts are reported. Data obtained with one of two distinct nontargeting (NT) or SETDB1 targeting shRNAs are shown. ( B ) H3K9me3 signal as a function of Hi-C compartment eigenvalue score. Shown are the H3K9me3 read counts per kilobase per compartment on the Y -axes compared with the compartment score on the X -axes in either compartment A (red) or compartment B (blue). Compartments were identified using Hi-C contact data at 40-kb resolution. ( C ) Mean read counts per kilobase in inactive ( left ) and active ( right ) compartments in control (NT sh; gray) and SETDB1-depleted (SETDB1 sh; orange) macrophages. Two-way ANOVA with Tukey multiple comparison test. (**) Adjusted P -value < 0.01. ( D ) H3K9me3 metaprofiles at genomic regions ( n = 7543) showing reduced H3K9me3 in SETDB1-depleted untreated macrophages. Shown is the metaplot of the replicate average of H3K9me3 signal per base pair in control (gray) and SETDB1-depleted (orange) conditions. ( E ) Cumulative megabases of genomic regions showing loss of H3K9me3 in SETDB1-depleted and untreated macrophages using different thresholds (adjusted P -value and fold change computed via DESeq2) for differential peak calling. ( F ) Effects of 2 h of LPS stimulation on the genomic profiles of H3K9me3 in untreated macrophages transduced with a nontargeting (NT) shRNA. ( G ) Effects of 2 h of LPS stimulation on the 7543 H3K9me3 blocks methylated by SETDB1. ( H ) H3K9me3 signal intensities at the 7543 H3K9me3 blocks methylated by SETDB1 in LPS-stimulated macrophages transduced with either a nontargeting (NT, X -axis) or a SETDB1 targeting ( Y -axis) shRNA.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Control, Derivative Assay, Hi-C, Comparison, Transduction, shRNA, Methylation

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Impact of SETDB1 depletion on the macrophage epigenome. ( A ) Genomic occupancy of PU.1 in control (NT sh; X -axis) and SETDB1-depleted (SETDB1 sh; Y -axis) untreated macrophages. PU.1 peaks were called using MACS2 with an FDR ≤10 −15 and retaining only peaks with overlap >50% in the two replicates ( n = 38,580). ( B ) Overlap between sites showing H3K9me3 signal loss in SETDB1-depleted macrophages and enhancers active in other cell types or tissues. ( C ) H3K27ac genomic distribution in control and SETDB1-depleted macrophages. ChIP-seq data sets were generated using cells transduced with two nontargeting (NT) or two SETDB1 targeting shRNAs. ( D ) Overlap of H3K27ac gain with genomic regions showing H3K9me3 reduction in SETDB1-depleted macrophages. ( E ) Overlap of genomic regions characterized by reduced H3K9me3 and gained H3K27ac in SETDB1-depleted macrophages with enhancers active in other lineages ( left ) or PU.1 peaks ( right ). ( F ) Genomic regions showing a gain in H3K27ac were divided based on the overlap with reduced H3K9me3 and analyzed for chromatin accessibility by ATAC-seq and PU.1 genomic occupancy. Shown is the metaplot of the replicate average of H3K27ac signal per base pair for the conditions reported. ( G ) Effects of LPS stimulation on H3K27ac at regions showing H3K27ac gain upon SETDB1 depletion. Shown are the box plots for the normalized reads per kilobase per individual replicates. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( H ) Overrepresentation of transcription factor motifs at genomic sites showing increased H3K27ac in SETDB1-depleted macrophages, divided based on the overlap with H3K9me3 loss. Enrichment was calculated using all accessible sites as background sequences.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Control, ChIP-sequencing, Generated, Transduction, Comparison

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: SETDB1 deposited H3K9me3 and control of CTCF recruitment to SINE B2 repeats. ( A ) Enrichment of different repeat families at genomic regions showing H3K9me3 loss in SETDB1-depleted macrophages. Reported is the Bonferroni adjusted P -value and the log 2 scaled ratio between expected and observed frequencies. ( B ) Motif overrepresentation analysis at repeats contained in genomic regions showing H3K9me3 reduction ( left ) or H3K9me3 loss and concurrent H3K27ac gain ( right ) upon SETDB1 depletion. Data are separately shown for LTR and SINE retrotransposons. Enrichment was computed using HOMER , providing distinct custom sets of background sequences: sequences of all LTRs and SINEs in the mm10 genome for the 7543 H3K9me3 set and sequences of LTRs and SINEs within the 7543 H3K9me3 sites for the 257 H3K9me3 sites with concurrent hyperacetylation. ( C ) CTCF genomic distribution in control and SETDB1-depleted macrophages. CTCF ChIP-seq was carried out using cells transduced with either two nontargeting (NT) shRNAs or two SETDB1 targeting shRNAs. ( D ) CTCF binding at genomic regions showing reduced H3K9me3 in SETDB1-depleted macrophages. ( E ) Quantitation of the effects shown in D reporting the normalized read counts per kilobase of CTCF in the various conditions. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( F ) CTCF peaks called as up-regulated (red), unaffected (gray), or down-regulated (blue) upon SETDB1 depletion. ( G ) Association of constitutive CTCF peaks and peaks induced upon SETDB1 depletion with different families of repeat elements. Shown is the repeat density per base pair. ( H ) Distance between CTCF peaks and H3K27ac peaks induced upon SETDB1 depletion. ( Left ) Distance between gained H3K27ac peaks ( n = 579) and constitutive (gray) or induced (red) CTCF peaks. (****) P < 0.0001, Mann–Whitney test. ( Right ) Bootstrap resampling test assessing the mean distance between randomly selected CTCF peaks and gained H3K27ac ( P < 0.0001, Wilcoxon signed rank test). Mean distance between induced CTCF peaks and up-regulated H3K27ac sites is shown with a dashed green line. ( I ) Cumulative distribution of the H3K9me3 signal centered on the summits of the CTCF peaks induced upon SETDB1 depletion. ( J ) Heat maps displaying CTCF and H3K9me3 ChIP-seq signals for constitutive and induced CTCF peaks in both control (NT sh) and SETDB1-depleted (SETDB1 sh) cells. Enlarged view for the 1644 induced CTCF peaks is shown at the right of each heat map. ( K ) Inverse relationship between CTCF binding intensity (±400 bp surrounding CTCF peak summit) and H3K9me3. CTCF peaks were divided into quintiles based on increasing H3K9me3 signal. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( L ) CpG DNA methylation at both CTCF-induced and not induced peaks. In case multiple CTCF motifs were associated with a single peak, only methylation data at the best motif were reported.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Control, ChIP-sequencing, Transduction, Binding Assay, Quantitation Assay, Comparison, MANN-WHITNEY, DNA Methylation Assay, Methylation

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Effect of SETDB1 depletion on chromatin organization in macrophages. ( A ) Insulation scores were measured at insulation sites in control (NT sh) and SETDB1-depleted (SETDB1 sh) macrophages, and differences were calculated. Insulation sites were identified genome-wide as point of local minima in the insulation score at 10-kb resolution. ( B ) CTCF peaks enhanced in SETDB1-depleted macrophages are associated with increased insulation capacity at pre-existing insulation sites. (**) P < 0.01, Mann–Whitney test. ( C ) Heat maps showing insulation scores in control macrophages (white/blue color palette) and the log 2 fold change (white/red color palette) between control and SETDB1-depleted macrophages (log 2 of the ratios between minimum–maximum normalized SETDB1 sh and NT sh bins) for genomic areas upstream of and downstream from the strand-oriented CTCF motifs. ( D ) Proportion of constitutive ( top ) and induced ( bottom ) CTCF peaks found at insulation sites. ( E ) Aggregate region analysis (ARA) showing Hi-C contact probabilities (i.e., average observed over expected contacts) in control (NT sh) and SETDB1-depleted (SETDB1 sh) macrophages. Plots are centered on either constitutive ( top ) or inducible ( bottom ) CTCF peaks matching insulation sites. Observed versus expected interaction ratios are shown together with the difference between control and SETDB1-depleted conditions. ( F ) Aggregate plots as in E centered on CTCF peaks elsewhere within TADs. ( G ) Frequency distribution ( top panel) of the differences in loop intensities normalized to local background ( bottom panel) in SETDB1-depleted versus control macrophages. ( H ) Aggregate plots ( left ) centered on down-regulated ( top ), invariant ( middle ), and up-regulated ( bottom ) loops in SETDB1-depleted versus control macrophages. ( I ) Box plots of the distribution of the fold changes of contact intensities per loop over the surrounding local background in control and SETDB1-depleted macrophages. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001, (**) P < 0.01, (*) P < 0.05. ( J ) Distance between induced CTCF peaks and induced loops. ( Left ) Distance between induced CTCF and invariant (gray), down-regulated (cyan), or induced (red) loop anchors. One-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( Right ) Bootstrap randomization test assessing the mean distance between randomly placed anchors and induced CTCF peaks ( P < 0.0001, Wilcoxon signed rank test). Mean distance between induced CTCF peaks and up-regulated loops is shown with a dashed green line.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Insulation, Control, Genome Wide, MANN-WHITNEY, Hi-C, Comparison

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Basal gene expression and its correlation with altered chromatin organization in SETDB1-depleted macrophages. ( A ) Heat map showing H3K27ac ChIP-seq signal in control macrophages (NT sh) at anchors of induced loops centered on the strongest H3K27ac peak. Each anchor pair was oriented in order to display the highest H3K27ac signal in the heat map at the right . The two heat maps were ranked based on the mean signal in anchors 1 and 2. ( B ) H3K27ac at induced loops is enhanced upon deletion of SETDB1. Wilcoxon matched-pairs signed rank test. (****) P < 0.0001. ( C ) Differentially expressed genes in untreated control and SETDB1-depleted macrophages. Data are from n = 2 independent biological replicates, each one done in duplicate with both shRNAs (total n = 4 samples per group). ( D ) Distance between up-regulated and down-regulated genes in SETDB1-depleted macrophages and induced CTCF peaks. (****) P < 0.0001, Mann–Whitney test. ( E ) Bootstrap randomization test assessing the mean distance between randomly placed anchors and up-regulated genes. The mean distance between induced loops and up-regulated genes is shown with a dashed green line. ( F ) Aggregate plots showing intensities of loops associated with genes up-regulated or down-regulated in SETDB1-depleted macrophages. ( Right ) Box plots showing the distribution of the fold changes of contact intensities per loop over the surrounding local background in control and SETDB1-depleted macrophages. Wilcoxon matched-pairs signed rank test. (****) P < 0.0001. ( G ) Genomic snapshot showing a genomic interval containing the Tmem154 gene. Data shown from top to bottom include H3K27ac, RNA-seq, contact density plot generated using Tmem154 as viewpoint (yellow highlight and red dashed square), loops identified via cLoop divided into up-regulated (red) and down-regulated (cyan), identified inducible CTCF peak upstream of Tmem154 (green), and normalized contact maps in control and SETDB1-depleted macrophages. ( H ) Zoomed-in view of the region surrounding Tmem154 showing H3K9me3 and CTCF peaks regulated by SETDB1 depletion. The M1-type and M2-type CTCF motifs contained in the SINE B2 elements are shown.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Gene Expression, ChIP-sequencing, Control, MANN-WHITNEY, RNA Sequencing, Generated

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Effects of SETDB1 depletion on LPS-regulated gene expression and its correlation with chromatin folding. ( A ) H3K27ac peaks significantly changed in LPS-stimulated macrophages (2 h). Up-regulated peaks are shown in red, and down-regulated peaks are shown in blue. Data are from n = 2 biological replicates. ( B , C ) Effects of SETDB1 depletion on LPS-regulated H3K27ac are shown as metaplots ( B ) or box plots of normalized and centered read counts per kilobase across all conditions ( C ). Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( D ) Distance between LPS-induced H3K27ac peaks and CTCF peaks induced upon SETDB1 depletion (expected distances were computed through 10,000 bootstrap resamplings of all CTCF peaks). One-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( E ) Gene expression changes induced by LPS stimulation (2 h) of mouse macrophages. ( F ) Effect of SETDB1 depletion on both LPS-induced (red) and down-regulated (blue) genes. ( G ) Quantification of the effects shown in F reporting the normalized and centered read counts per kilobase across all conditions. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( H ) Representative snapshots of LPS-inducible and SETDB1-regulated genes. ( I ) Distance between LPS-regulated genes and CTCF peaks induced in SETDB1-depleted macrophages (expected distances were computed through bootstrap resampling of all CTCF peaks). One-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( J ) Genomic snapshot showing the LPS-inducible Egr2 gene. The heat maps display normalized contact densities in control ( top ) and SETDB1-depleted ( bottom ) macrophages. The contact density plot generated using Egr2 as the viewpoint is shown together with the position of induced (red) and constitutive (gray) CTCF peaks.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Gene Expression, Comparison, Control, Generated