Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 1. A genome-wide CRISPR-Cas9 genetic screen identifies an essential requirement for CRAMP1 and histone H1.4 in PRC2-mediated reporter repression (A) Schematic representation of GFP reporter repression by the PRC2 complex. (B) The GFP reporter is derepressed upon CRISPR-Cas9-mediated gene disruption of any of the three core PRC2 subunits, as assayed by flow cytometry. (C) A genome-wide CRISPR-Cas9 screen to identify factors required for PRC2 function. Following Cas9 expression in KBM-7 cells harboring the PRC2-sensitive GFP reporter, genome-wide mutagenesis was carried out with the Sabatini/Lander single guide RNA (sgRNA) library, 36 and GFP + cells isolated through two sequential rounds of FACS. ‘‘Significance’’ on the y axis represents the negative log of the ‘‘pos|score’’ metric reported by Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK). 37

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: Genome Wide, CRISPR, Disruption, Flow Cytometry, Expressing, Mutagenesis, Isolation, Knock-Out

Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 5. Linker histones are not enriched at regions marked by H3K9me3 (A–D) Lack of linker histone enrichment at H3K9me3-marked genomic regions. (A) Tornado plots depicting linker histone CUT&Tag signal across H3K9me3 peaks from the ENCODE project; average signal intensity is shown in (B). (C) Heatmap depicting the lack of correlation between linker histone occupancy and H3K9me3. Cells are annotated with pairwise Spearman correlation coefficients. An example locus is shown in (D). (E) CUT&Tag faithfully profiles H3K9me3. Example loci comparing CUT&Tag versus H3K9me3 ChIP-seq data (ENCODE) are shown. (F and G) Linker histone insufficiency does not impair H3K9me3-dependent LINE-1 silencing by the HUSH complex. (F) Schematic representation of the dual- color reporter cell line designed to monitor both H3K9me3-dependent repression by the HUSH complex and linker histone-mediated PRC2-reporter repression. (G) HUSH-mediated LINE-1 silencing is unaffected upon CRAMP1 depletion. The indicated CRISPR sgRNAs were expressed in the dual-color reporter cell line, and GFP and iRFP fluorescence assayed by flow cytometry. See also Figure S5 and Table S2.

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: ChIP-sequencing, CRISPR, Fluorescence, Flow Cytometry

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Establishment of the DEPDC5-KO HCC cells by using CRISPR/Cas9 system. ( a ) Schematics of the protein structure of DEPDC5. Grey and black bars show the position of amino acid substitutions induced by missense and stop-gain mutations in the ICGC public data. The arrow indicates the site that an sgRNA targets for knockout by using CRISPR/Cas9 technology in this study. ( b ) Sequence chromatograms of the DEPDC5-KO JHH5 and HLE cells around the sgRNA target site (grey background color). ( c ) Immunofluorescence analysis of the DEPDC5-WT and -KO JHH5 and HLE cells with DEPDC5 staining (red). Nuclei were counterstained with DAPI (blue). Magnification, ×200.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: CRISPR, Knock-Out, Sequencing, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Cellular response of the DEPDC5-KO cells to leucine deprivation involved in autophagy pathway. ( a ) Proliferation curves of the DEPDC5-WT and -KO JHH5 and HLE cells. The value of each sample was relative to that at Day 1. Error bars are the mean ± S.D. P values were calculated by Welch’s t -test. ( b ) Flow cytometric analysis with PI staining. The percentage of each phase is the mean ± S.D. P values were calculated by Welch’s t -test. ( c ) Immunoblots of LC3B and p62. The cells were exposed to leucine-free medium for the indicated time periods. GAPDH was used as a loading control. ( d ) Immunofluorescence analysis of the DEPDC5-WT and -KO JHH5 and HLE cells under leucine-depleted conditions with LC3 (green) and p62 (red) staining. Nuclei were counterstained with DAPI (blue). Magnification, ×200.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Staining, Western Blot, Control, Immunofluorescence

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Reduction of cellular ROS levels in the DEPDC5-KO HCC cells. ( a ) Representative histogram images of cells with CellROX. The concentration of H2O2 was 100 μM in the right panels. The value of each mean relative fluorescence intensity (RFI) is the mean ± S.D. P values were calculated by Welch’s t -test. ( b ) Dose-response curves of the cell viability after H2O2 treatment. P values were calculated from the ANOVA table.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Concentration Assay, Fluorescence

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Inhibition of cancer cell growth with elevated cellular ROS levels by DEPDC5 overexpression. ( a ) Immunofluorescence analysis of the doxycycline (DOX)-inducible DEPDC5-expressing HuH7 cells with DEPDC5 staining (red). Nuclei were counterstained with DAPI (blue). Magnification, ×200. ( b ) Quantification of colony-forming efficiency. Error bars are the mean ± S.D. P values were calculated by Welch’s t -test. ( c ) Immunoblots of p62. The cells were exposed to medium containing doxycycline for the indicated time periods. GAPDH was used as a loading control. ( d ) Representative histogram images of cells with CellROX. The value of each mean RFI is the mean ± S.D. P values were calculated by Welch’s t -test. ( e ) In vivo tumorigenicity of doxycycline-inducible DEPDC5-expressing HCC cells. The upper and lower panels show growth curves of transplanted tumors and representative photo images. Error bars are the mean ± S.E in the upper panel. P values were calculated by Welch’s t -test. The white scale bar is 1 cm in the lower panel.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Inhibition, Over Expression, Immunofluorescence, Expressing, Staining, Western Blot, Control, In Vivo

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Relationship among DEPDC5 and p62 expression in HCC samples and patient prognosis. ( a ) Immunohistochemical analysis of DEPDC5 and p62 in a representative tissue sample including adjacent liver tissue (N) and cancer (T). Nuclei were counterstained with hematoxylin. In adjacent liver tissues of almost all cases, DEPDC5 was positive while p62 was negative. ( b ) Kaplan-Meier curves of the progression-free and overall survival in groups of HCC patients classified according to DEPDC5 and p62 expression patterns. P values were calculated by the log-rank test.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Expressing, Immunohistochemical staining

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Relationship between DEPDC 5 expression and clinicopathological factors.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques: Expressing

Journal: Scientific Reports

Article Title: DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma

doi: 10.1038/s41598-017-18323-9

Figure Lengend Snippet: Univariate and multivariate analysis of factors contributing to progression-free and overall survival.

Article Snippet: The entire coding sequences of DEPDC5 and eGFP were amplified by using the primer pair sets, 5′-ATGAATCGATATGAGAACAACAAAGGTCTACAAAC-3′ (forward) and 5′-CTCCTCGCCCTTGCTCACCATCGGGGCACTGGCATGCATC-3′ (reverse) for DEPDC5, and 5′-GATGCATGCCAGTGCCCCGATGGTGAGCAAGGGCGAGGAG-3′ (forward) and 5′-TATAGCGGCCGCTTACTTGTACAGCTCGTC-3′ (reverse) for eGFP from eGFP Depdc5 pLJM1 vector (Addgene, plasmid #46380; a kind gift from Dr. David Sabatini).

Techniques:

Journal: Scientific Reports

Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

doi: 10.1038/srep42127

Figure Lengend Snippet: ( A ) Cultured primary HLMVECs were transduced by EGFP-Cas9 adenovirus and sgRNA lentivirus targeting on Tie2 and subjected to flow cytometry analysis of EGFP expression. In a separate study, HLMVECs were transduced by GFP lentivirus and subjected to flow cytometry analysis of EGFP expression as an indicator of lentivirus transduction efficiency. ( B ) Protein expression of Tie2 in vector control and Tie2 knockout HLMVECs induced by CRISPR-Cas9 was determined by immunoblotting. Overexpression of mouse Tie2 in Tie2 −/− HLMVECs was determined to assess the ability to rescue Tie2 expression after deletion. The uncropped full-length gels can be found in . ( C ) Quantification of Tie2 protein expression from 3 independent experiments. sgRNA Tie2-1 and sgRNA Tie2-2 are CRISPR-Cas9-mediated deletions of Tie-2 at two distinction domains of Tie2 (two different sgRNAs). HuTie2KO1 and HuTie2KO2 + mTie2 represent restoration of Tie-2 expression in cells having undergone Tie-2 deletion. Differences were calculated using one-way ANOVA. P values less than 0.05 are indicated in the graph. ( D ) T7E1 assay detecting mutation on HLMVECs edited by CRISPR-Cas9 with Tie2 sgRNAs as showing a series of bands. Wild-types cells with or without Cas9 or vector only show single bands (negative controls).

Article Snippet: DNA oligos were synthesized by IDT and the overlapping PCR products were cloned into pLX-single sgRNA lentiviral vector (a gift from Eric Lander and David Sabatini and obtained through Addgene as plasmid # 50662).

Techniques: Cell Culture, Flow Cytometry, Expressing, Transduction, Plasmid Preparation, Control, Knock-Out, CRISPR, Western Blot, Over Expression, Mutagenesis

Journal: Scientific Reports

Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

doi: 10.1038/srep42127

Figure Lengend Snippet: ( A ) Indel size distribution of wild-type cells (control) and cells transduced with two different sgRNAs targeting Tie2 (sgRNA Tie2-1, sgRNA Tie2-2). ( B ) NHEJ reads with insertions, deletions, and substitutions were mapped to reference amplicon position. Sequencing/alignment errors (green lines) can be distinguished from indels by their similar positions in all three samples. ( C ) Frameshift mutagenesis profile and predicted Cas9 cleavage site. Unmodified reads are excluded from this analysis. sgRNA Tie2-1 and sgRNA Tie2-2 showed different percentage of reads with mutations from frameshift, in frame, and in noncoding region. ( D ) Predicted impacts on splice sites. Potential splice sites modified refers to the reads in which either of the two introns adjacent to the exon is disrupted. ( E ) The relative contributions of potentially disruptive coding region mutations (indels) and non-coding region mutations (5′ splice site) were quantified across 3 passages. SNPs, in-frame indels, indels less than 5 residues, and 3′ splice site mutations were excluded.

Article Snippet: DNA oligos were synthesized by IDT and the overlapping PCR products were cloned into pLX-single sgRNA lentiviral vector (a gift from Eric Lander and David Sabatini and obtained through Addgene as plasmid # 50662).

Techniques: Control, Transduction, Amplification, Sequencing, Mutagenesis, Modification

Journal: Scientific Reports

Article Title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

doi: 10.1038/srep42127

Figure Lengend Snippet: ( A ) Basal TER and post-thrombin (1 U/ml) TER were studied in confluent control, Tie2-deleted HLMVECs and mTie2 overexpressing cells in which Tie2 had been deleted. Absolute TER values were reduced in both Tie2-deleted groups as compared to control ECs at basal condition. mTie2 overexpression successfully rescued the basal leakiness. ( B ) Quantification of TER values of wild-type (control), transduced cells (sgRNA Tie2-1, sgRNA Tie2-2) and rescued cells (HuTie2KO + mTie2) at basal (−1 h), thrombin-stimulated (0.5 h) and post-recovery (3 h) condition. Differences were calculated using two-way ANOVA. P values less than 0.05 are indicated in the graph. n = 3. ( C ) Serum-starved confluent control or Tie2-deleted HLMVECs were challenged by PBS or 1 U/ml of thrombin, and subjected for VE-cadherin immunostaining at the indicated time-points and analyzed by confocal microscopy. The marked disruption of VE-cadherin junctions seen in wild-type HLMVEC monolayer (control) at the 30 min post thrombin (white arrows) was reversed by 2 h; however, the defective VE-cadherin junctions were present in Tie2-deleted HLMVECs 2 h post-thrombin. White arrows are used to identify areas of adherens junction disruption where neighboring cells lack cell membrane localization of VE-cadherin. Results are representative of 3 independent experiments.

Article Snippet: DNA oligos were synthesized by IDT and the overlapping PCR products were cloned into pLX-single sgRNA lentiviral vector (a gift from Eric Lander and David Sabatini and obtained through Addgene as plasmid # 50662).

Techniques: Control, Over Expression, Immunostaining, Confocal Microscopy, Disruption, Membrane

Journal: bioRxiv

Article Title: Inhibition of mitochondrial ferredoxin 1 (FDX1) prevents adaptation to proteotoxic stress

doi: 10.1101/288365

Figure Lengend Snippet: (A) Schematic depicting the pooled CRISPR-based screen. (B) Gene scores in elesclomol-1-(100 nM) and elesclomol-2-(1uM) treated K562 cells. The gene score is the median log2 fold change in abundance of all sgRNAs targeting that gene during the culture period. The FDX1 score is indicated. (C) The corrected p-values (-log10) of the KS tests of the sgRNA distribution for each gene vs the distribution of all sgRNAs in the screen in the eleslcomol-1 and elesclomol-2 screens. Values are ordered on the x-axis by chromosome and location; the dotted line indicates a corrected p-value of 0.05. The FDX1 score is indicated. (D) Western blot analysis of FDX1 and tubulin (loading control) protein expression levels in WT K562 cells (WT) or cells with FDX1 (two distinct sgRNAs) and AAVS1 deletions. (E-F) Viability curves of parental K562 cells and cells deleted for either AAVS1 (control) or FDX1 achieved with two sgRNAs using CRISPR/Cas9. (E) The indicated cells were treated with increasing concentrations of eleslcomol-1 and viability was examined after 72 hours. (F) The indicated cells were grown in the presence of either glucose or galactose and the relative cell number plotted.

Article Snippet: 268M cells were transduced with 22mL human genome-wide cleavage-optimized lentiviral sgRNA library containing Cas9 ( http://www.addgene.org/pooled-library/sabatini-crispr-human-high-activity-3-sublibraries ) as previously described ( ).

Techniques: CRISPR, Western Blot, Expressing