sgrna library targets  (Synthego Inc)

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A . Identification of host restriction genes from the CRISPR knockout screen. The A549 cell library containing genome-wide CRISPR knockout sgRNAs were infected with HCoV-229E-mGreen (MOI 0.5, 24 h). Infected reporter-positive cells were sorted for genomic DNA extraction and subsequent sgRNA sequence analysis. Genes were analyzed using MAGeCK software and ranked based on -log10 (MAGeCK score) and P -values. The algorithm employs one-sided test to identify genes under positive selection, and P -values were adjusted for multiple testing using the Benjamini-Hochberg method. B Validation of the 12 top-ranked genes, with a cutoff of false discovery rate (FDR) < 0.05. A549 cells were edited with two independent sgRNAs per gene. Following infection with HCoV-229E (MOI 0.5, 24 h), infection efficiency was quantified by flow cytometry for the percentage of nucleocapsid ( N )-positive cells. C Representative images of control and UHRF1 -knockout A549 cells infected with HCoV-229E (MOI 0.5, 24 h), and analyzed by Operetta high-content imaging from three independent experiments. Scale bar, 100 μm. D Cell viability of control and gene-knockout A549 cells was assessed 48 h after seeding. E Overexpression of human UHRF1 inhibits HCoV-229E infection. HeLa cells overexpressing UHRF1 were infected with HCoV-229E (MOI 0.5, 24 h). Virus infection efficiency was determined by flow cytometry. F Trans-complementation of UHRF1 in knockout cells inhibits HCoV-229E infection. A549 cells with or without exogenous UHRF1 expression were edited with sgRNA targeting endogenous UHRF1, and infected with HCoV-229E (MOI 0.5, 24 h). Error bars represent standard deviations from three independent experiments ( n = 3), and each performed in duplicate. Two-way ANOVA with Sidak’s test ( B , F ); unpaired, two-sided t-test ( D , E ); mean ± s.d.; * P < 0.05; *** P < 0.001; **** P < 0.0001; ns not significant.

Article Snippet: Briefly, A549 cells expressing the Cas9 (A549-Cas9) were transduced with a packaged sgRNA lentivirus library targeting 19,114 genes (Addgene #73178) , at a multiplicity of infection (MOI) of ~0.3 by spinoculation at 1000 x g and 32 °C for 30 min in 12-well plates.

Techniques: CRISPR, Knock-Out, Genome Wide, Infection, DNA Extraction, Sequencing, Software, Selection, Biomarker Discovery, Flow Cytometry, Control, Imaging, Gene Knockout, Over Expression, Virus, Expressing

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A –C Pseudovirus infection assays. Control and UHRF1 -knockout A549-ACE2 cells were infected with VSV-based pseudoviruses. D –F Virus binding and internalization assays. Cells were incubated with HCoV-229E (MOI 10). Bound or internalized virions were quantified by qRT-PCR or analyzed by confocal microscopy. Representative images from three independent experiments were shown. G , H Western blotting analysis of gene expression in gene-knockout A549 or HeLa cells. Representative images from three independent experiments were shown. I Infection efficiency of HCoV-229E (MOI 0.5, 24 h) in control and UHRF1 -knockout HeLa cells, determined by flow cytometry. J . Relative APN mRNA levels and infection efficiency of HCoV-229E (MOI 1, 12 h) in control and UHRF1 -knockout primary human bronchial epithelial cells (HBEC). mRNA levels were analyzed by qRT-PCR, and infectivity was determined by flow cytometry. K Temporal expression of APN in UHRF1 -knockout A549 cells. Cell lysates were collected at different days post-transduction of sgRNA-expressing lentivirus and analyzed by western blotting. Representative images from three independent experiments were shown. L Surface expression of APN analyzed by flow cytometry in A549 cells edited with control or UHRF1 sgRNA. M Relative APN mRNA levels analyzed by qRT-PCR in A549 cells edited with control or UHRF1 sgRNA. N Relative APN mRNA levels analyzed by qRT-PCR in A549 cells treated with UHRF1 inhibitor UF146 for 2 days. O Control and two APN -knockout A549 clonal cell lines were edited with control or UHRF1 sgRNA, and infected with HCoV-229E (MOI 0.5, 24 h). Infectivity was determined by flow cytometry. P UHRF1 -knockout cells were pre-treated with 5 μg/ml APN-blocking antibody or isotype control for 1 h, then infected with HCoV-229E (MOI 0.5, 24 h) in the presence of antibody. qRT-PCR was performed to determine the relative levels of HCoV-229E N gene. Error bars represent standard deviations from three independent experiments ( n = 3), and each performed in duplicate. Unpaired, two-sided t-test ( A – C , I , J , M ); two-way ANOVA with Sidak’s test ( D, O ); one-way ANOVA with Sidak’s test ( N , P ); mean ± s.d.; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Briefly, A549 cells expressing the Cas9 (A549-Cas9) were transduced with a packaged sgRNA lentivirus library targeting 19,114 genes (Addgene #73178) , at a multiplicity of infection (MOI) of ~0.3 by spinoculation at 1000 x g and 32 °C for 30 min in 12-well plates.

Techniques: Infection, Control, Knock-Out, Virus, Binding Assay, Incubation, Quantitative RT-PCR, Confocal Microscopy, Western Blot, Gene Expression, Gene Knockout, Flow Cytometry, Expressing, Transduction, Blocking Assay

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A UHRF1 does not regulate the expression of exogenous HA-tagged APN and ACE2 from transiently transfected plasmids. Representative Western blotting images from three independent experiments were shown. B Bisulfite sequencing of CpG sites in APN proximal promoter from control and UHRF1 -knockout A549 cells. The methylation (Meth) rate was calculated as the ratio of methylated sites to the total number of sites tested. C A549 cells were treated with 5-AZA for 3 days, and relative APN mRNA levels were determined by qRT-PCR. D , E Huh7 stably expressing DNMT3A were generated by lentivirus transduction. Relative APN mRNA levels were determined by qRT-PCR ( D ), and infectivity was detected by flow cytometry after infection with HCoV-229E (MOI 0.01, 24 h) ( E ). F , G In vitro methylation and dual-luciferase reporter assays. The methylation status of luciferase reporter plasmid was verified by HpaII/MspI digestion ( F ). Representative image from three independent experiments was shown ( F ). The luciferase activity of unmethylated (Unmeth) or methylated (Meth) luciferase reporter plasmid co-transfected with internal control pRL-TK was measured at 24 h post-transfection. Results were normalized to the unmethylated plasmid ( G ). H Electrophoretic mobility shift assay (EMSA). Nuclear extracts were incubated with biotin-labeled unmethylated or methylated APN promoter probe to detect DNA-protein complexes. Representative images from three independent experiments were shown. I , J Chromatin immunoprecipitation (ChIP) assay with c-Maf expression. qPCR was performed to detect c-Maf binding to the transcription factor (TF) binding site ( I ) or CpG island ( J ) of the APN proximal promoter. K Schematic diagram of UHRF1 truncations. L , M UHRF1 -knockout A549 stably expressing wild-type or truncated UHRF1 were established by lentivirus transduction and verified by western blotting ( L ). Representative images from three independent experiments were shown ( L ). Cells were infected with HCoV-229E (MOI 0.5, 24 h) at day 10 post-transduction, and the infectivity was determined by flow cytometry ( M ). Error bars represent standard deviations from three independent experiments ( n = 3), and each performed in duplicate. One-way ANOVA with Sidak’s test ( C , M ); unpaired, two-sided t-test ( D , E , G , I – J ); mean ± s.d.; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Briefly, A549 cells expressing the Cas9 (A549-Cas9) were transduced with a packaged sgRNA lentivirus library targeting 19,114 genes (Addgene #73178) , at a multiplicity of infection (MOI) of ~0.3 by spinoculation at 1000 x g and 32 °C for 30 min in 12-well plates.

Techniques: Expressing, Transfection, Western Blot, Methylation Sequencing, Control, Knock-Out, Methylation, Quantitative RT-PCR, Stable Transfection, Generated, Transduction, Infection, Flow Cytometry, In Vitro, Luciferase, Plasmid Preparation, Activity Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Chromatin Immunoprecipitation, Binding Assay

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A Volcano plot of RNA-seq analysis. Total cellular RNA was extracted from control and UHRF1 -knockout A549-ACE2 cells and subjected to RNA-seq. Genes with an absolute Log 2 fold change >2 and adjusted P -value < 0.05 were considered as differentially expressed. Differential expression analysis was performed using DESeq2 with a two-sided Wald test. P -values were adjusted for multiple comparisons using the Benjamini-Hochberg method. B Schematic of focused CRISPR activation screening. A sub-library targeting 2172 of the 2210 upregulated genes identified from RNA-seq analysis of UHRF1 -knockout cells, with ~4 sgRNAs per gene, was generated and transduced into A549-ACE2-dCas9 cells. Cells were infected with HCoV-229E-mGreen (MOI 0.5, 24 h) or SARS-CoV-2 transcription- and replication-competent virus-like particles in which the N gene is replaced by the reporter GFP (trVLP-GFP) (MOI 0.5, 24 h). Infected reporter-positive cells were sorted for genomic DNA extraction and sgRNA sequence analysis. Created in BioRender. Wang, P. (2025) https://BioRender.com/35yt09k . C , D Genes identified from CRISPR screens for HCoV-229E ( C ) and SARS-CoV-2 ( D ). Genes were analyzed by MAGeCK software and sorted based on -log 10 (MAGeCK score) and P -values. The algorithm employs a one-sided test to identify genes under positive selection, and P -values were adjusted for multiple testing using the Benjamini-Hochberg method.

Article Snippet: Briefly, A549 cells expressing the Cas9 (A549-Cas9) were transduced with a packaged sgRNA lentivirus library targeting 19,114 genes (Addgene #73178) , at a multiplicity of infection (MOI) of ~0.3 by spinoculation at 1000 x g and 32 °C for 30 min in 12-well plates.

Techniques: RNA Sequencing, Control, Knock-Out, Quantitative Proteomics, CRISPR, Activation Assay, Generated, Infection, Virus, DNA Extraction, Sequencing, Software, Selection

Journal: Nature Communications

Article Title: PTPN23-dependent ESCRT machinery functions as a cell death checkpoint

doi: 10.1038/s41467-024-54749-2

Figure Lengend Snippet: a Gene essentiality of mouse PTPs in RN2 cells and NIH 3T3 cells. sgRNA frequencies at Day 2 and Day 14 were determined by next-generation sequencing, with average logarithmic frequency changes calculated. Yellow dots represent pan-essential genes spiked into the screen library. b Histogram of PTPN23 gene knockout effect in 1078 cancer cell lines (DepMap 22Q2). The dashed line indicates the median score. c GFP competition growth assays performed in RN2 cells ( n = 3 independent experiments). d Left, Human PTPN23 and ALIX full-length constructs and PTPN23 truncations used in rescue experiments. Right, Heatmap visualizing the complementation effects aligned with the corresponding numbers on D14 in ( e ). His: His domain; PRR: Proline-rich region, located at the N-terminus of the His domain. e GFP competition growth assays performed in RN2 stable cell lines expressing indicated cDNAs. Top, control sgRNAs; bottom, PTPN23 sgRNAs ( n = 3 independent experiments). f Left, immunoblot of NOMO-1 cells overexpressing empty vector (EV), sgRNA-sensitive (PTPN23-WT) or sgRNA-resistant (PTPN23-r2) PTPN23 cDNA ( n = 3 independent experiments). Right, GFP competition growth assays performed using NOMO-1 stable cell lines expressing either PTPN23-WT ( n = 3 independent experiments) or PTPN23-r2 ( n = 4 independent experiments). g Top, acute depletion of PTPN23 with dTAG system. NOMO-1 cells stably expressing sgRNA-resistant PTPN23 fused with dTAG and endogenous PTPN23 were depleted with two sgRNAs. Bottom, immunoblotting analysis of PTPN23 examined after 4-day treatment of dTAG-13 (100 nM). h CellTiter-Glo (CTG) luminescence cell viability assay measured on cells treated with either DMSO or dTAG-13. DMSO or 500 nM dTAG-13 were added to three PTPN23-dTAG NOMO-1 cell lines established in ( g ) ( n = 3 independent experiments). i Cell death indicated by Sytox Green positivity. PTPN23-dTAG NOMO-1 cells were treated with 200 nM dTAG-13 for 4 days, and stained with Sytox Green ( n = 60 fields captured, 3 independent experiments). Scale bar: 30 μm. Data are presented as mean ± SEM, statistical analysis for ( h ) by Two-way ANOVA, Sidak’s multiple comparisons test; ( i ) by One-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: The pooled library containing phosphatase domain targeting and control sgRNAs (Supplementary Data ) was synthesized in duplicates or triplicates on an array platform (Twist Bioscience) and then cloned into the LRG2.1 vector with Gibson Assembly kit (NEB).

Techniques: Next-Generation Sequencing, Gene Knockout, Construct, Stable Transfection, Expressing, Control, Western Blot, Plasmid Preparation, Viability Assay, Staining

Journal: Nature Cell Biology

Article Title: Defining E3 ligase–substrate relationships through multiplex CRISPR screening

doi: 10.1038/s41556-023-01229-2

Figure Lengend Snippet: a-d Derivation of cells expressing C-terminal peptides targeted by Cul2 complex or Cul4 complexes. Starting from HEK-293T cells expressing the C-terminome GPS-peptide library , we isolated the most unstable GFP-peptide fusions (Bin1) (a) , treated them with MLN4924, and then isolated cells in which the GFP-peptides fusions were stabilized (b) . After recovery in the absence of MLN4924, to further purify substrates of specific Cullin complexes we expressed dominant-negative (DN) versions of either Cul2 (green) or Cul4A (pink) and again isolated the cells in which the GFP-peptides fusions were stabilized by FACS (c) . After recovery, we re-challenged the sorted cells with the DN Cullins to verify that the final populations of cells were highly enriched for CRL substrates (d) . e,f , Generation of the multiplex CRISPR screening vector to examine C-terminal degrons targeted by Cullins. e , Schematic representation of the multiplex CRISPR screening vector. GFP-fusion peptides were amplified from the genomic DNA of the cells in (d) by PCR and cloned into the lentiviral vector downstream of GFP; the CRISPR sgRNA library targeting either Cul2 or Cul4 adaptors was then cloned into the resulting substrate library using the I-SceI site to generate the dual GPS-sgRNA multiplex CRISPR screening library. f , The library was then introduced into HEK-293T stably expressing Cas9, and the top ~5% of cells expressing the most stable substrates were isolated by FACS. Genomic DNA was then extracted from both the sorted cells and the unsorted libraries, and substrate-sgRNA pairs enriched in the sorted cells quantified by paired-end Illumina sequencing.

Article Snippet: A custom sgRNA library targeting either Cul2/5 adaptors or Cul4 adaptors (six sgRNAs per gene) was synthesized as an oligonucleotide pool (Twist Bioscience), amplified by PCR (Q5 Hot Start High-Fidelity DNA Polymerase, NEB), purified (Qiagen PCR purification kit) and digested with BbsI (NEB).

Techniques: Expressing, Isolation, Dominant Negative Mutation, Multiplex Assay, CRISPR, Plasmid Preparation, Amplification, Clone Assay, Stable Transfection, Sequencing

Journal: Nature Cell Biology

Article Title: Defining E3 ligase–substrate relationships through multiplex CRISPR screening

doi: 10.1038/s41556-023-01229-2

Figure Lengend Snippet: a , Schematic representation of the dual GPS/CRISPR multiplex screening library, in which the GFP-fusion substrates were a pool of peptides enriched for C-terminal degrons targeted by Cul2 or Cul4 E3 ligase complexes, and the CRISPR sgRNA library targeted either Cul2/5 or Cul4 adaptors. b , c , Identification of KLHDC2 substrates bearing C-terminal di-glycine motifs: the multiplex screen results for six example substrates, all of which terminate with two glycine residues ( b ); the performance of sgRNAs targeting KLHDC2 across all substrates ( c ). d , e , Cullin adaptors are correctly assigned to their cognate C-terminal degrons. A range of peptide substrates bearing canonical C-degron motifs targeted by Cul2 ( d ) and Cul4 ( e ) adaptors were successfully identified. All source numerical data are available in Supplementary Tables – .

Article Snippet: A custom sgRNA library targeting either Cul2/5 adaptors or Cul4 adaptors (six sgRNAs per gene) was synthesized as an oligonucleotide pool (Twist Bioscience), amplified by PCR (Q5 Hot Start High-Fidelity DNA Polymerase, NEB), purified (Qiagen PCR purification kit) and digested with BbsI (NEB).

Techniques: CRISPR, Multiplex Assay