Journal: Nature Communications

Article Title: Orthogonal and multiplexable genetic perturbations with an engineered prime editor and a diverse RNA array

doi: 10.1038/s41467-024-55134-9

Figure Lengend Snippet: a Illustration of gene repression using PE2-mediated CRISPRi strategy targeting an EGFP reporter gene. b EGFP reporter repression with 33 different sgRNAs that span the EGFP gene. c Schematic of gene silencing achieved via shRNAs produced by the DAP array. d Comparative analysis of gene repression efficiencies using three different methods: PE2-mediated CRISPRi, dCas9-KRAB-MECP2 fusion protein, and DAP shRNA-mediated RNAi. GPP: Broad Institute GPP web portal. e Repression of the endogenous MLH1 gene using shRNAs designed by different web tools and expressed from DAP arrays. GEN GenScript siRNA design tool, INV InvivoGen siRNA Wizard . f Multiplex gene repression with DAP-shRNA array. FWD and REV are two DAP arrays encoding the same set of shRNAs in opposite order. Experiments were performed in HEK293T cells and analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or flow cytometry. Bars represent mean ± s.d. from n = 4 ( b , d ) and n = 3 ( e , f ) independent biological replicates. Source data are provided as a file.

Article Snippet: GEN GenScript siRNA design tool, INV InvivoGen siRNA Wizard . f Multiplex gene repression with DAP-shRNA array.

Techniques: Produced, shRNA, Multiplex Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry

Journal: Nature Communications

Article Title: Orthogonal and multiplexable genetic perturbations with an engineered prime editor and a diverse RNA array

doi: 10.1038/s41467-024-55134-9

Figure Lengend Snippet: a Schematic of a hypothetical complex genetic disease model involving Wilson’s disease, Type I diabetes, and Transthyretin amyloidosis. Treatment of the disease model requires orthogonal editing of the ATP7B gene, activation of the PDX1 gene, and repression of the TTR gene. b Design of a DAP array encoding a shRNA for gene silencing, a truncated agRNA for gene activation, and a ngRNA and a epegRNA for gene editing. c , d Therapeutic genetic perturbation in HepG2 disease cell line transfected by plasmids encoding the DAP array, PEAK, and MPH. REV: the direction of DAP array was reversed as compared to FWD DAP array. e Genetic perturbation in HEK293T cells transfected with plasmids encoding the DAP array, PEAK, and MPH to install the c.3207C>A mutation in the ATP7B gene, upregulate the expression of RHOXF2 gene, and silence the MLH1 gene. f , g Combinatorial delivery of the DAP array (AAV), PEAK (mRNA), and MPH (mRNA) into HEK293T cells. h , i Combinatorial delivery using plasmids for the DAP array and MPH, and lentivirus for PEAK. Controls were untreated cells. A stable cell line expressing PEAK was established before introducing the DAP array and MPH via plasmid transfection. Gene editing outcomes were analyzed by Sanger sequencing and transcriptional regulations were analyzed by RT-qPCR. Error bars represent mean ± s.d. from n = 3 independent biological replicates. Source data are provided as a file. c , f , h Created in BioRender. Yuan, Q. (2023) BioRender.com/b09r397.

Article Snippet: GEN GenScript siRNA design tool, INV InvivoGen siRNA Wizard . f Multiplex gene repression with DAP-shRNA array.

Techniques: Activation Assay, shRNA, Transfection, Mutagenesis, Expressing, Stable Transfection, Plasmid Preparation, Sequencing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Linker histone H1-0 is a specific mediator of the repressive ETV6::RUNX1 transcriptional landscape

doi: 10.1101/2024.06.28.601221

Figure Lengend Snippet: (A) H1-0 expression determined by RT-qPCR and representative Western Blot of REH cells treated for 48 hours with a non-targeting siRNA pool (siCtrl) or H1-0 -targeting siRNA pools siH1-0_1 or siH1- 0_2. Data is presented as the mean ± standard deviation. (B) Enrichment map of gene sets enriched in siCtrl REH cells compared to siRNA-mediated knockdown of H1-0 (cut-offs: p<0.005, false discovery rate (FDR) q-value<0.1) using the canonical pathways gene set collection (Human MSigDB Collections). No significantly enriched gene sets were found in siH1-0 REH cells using the indicated cut-offs. Groups of similar pathways are indicated. (C) Ingenuity Pathway analysis (IPA, QIAGEN) of upstream regulators significantly enriched in both siH1-0_1 versus siCtrl and siH1-0_2 versus siCtrl (p<0.05). (D) GSEA results of siH1-0 versus siCtrl using a published geneset of 103 significantly upregulated genes in both REH and AT-2 cells upon ETV6::RUNX1 knockdown (cut-offs: log2 fold change >0.9 and adjusted p<0.05). Normalized enrichment score (NES) and FDR are indicated. (E) GSEA of siH1-0 versus siCtrl using the HALLMARK_P53_PATHWAY gene set derived from Human MSigDB Collections. (F) RNA expression levels of EPOR , RAG1 and MDM2 determined by RNA-seq in siCtrl and siH1-0 REH. (G) Pearson correlation of H1-0 expression with EPOR or RAG1 expression in ETV6::RUNX1 + BCP-ALL patient samples derived from the PeCan St. Jude cloud (n=87, ( ; )).

Article Snippet: Specific siRNA sequences for knockdown of H1-0 in REH cells were designed using the Eurofins siRNA design tool ( https://eurofinsgenomics.eu/en/ecom/tools/sirna-design/ ) and purchased from Eurofins Genomics.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Knockdown, Derivative Assay, RNA Expression, RNA Sequencing Assay

Journal: Medicines

Article Title: Triple Silencing of HSP27, cFLIP, and CLU Genes Promotes the Sensitivity of Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells

doi: 10.3390/medicines11030007

Figure Lengend Snippet: Gene and protein expressions after the siRNA silencing of HSP27, cFLIP, and CLU genes in PC-3 cells. The efficiency of siRNA knockdown was verified by analyzing mRNA and protein levels by qRT-PCR and Western blotting, respectively. ( A ) After the siRNA treatment, the mRNA expression of HSP27, cFLIP, and CLU was significantly reduced. Data represent the mean ± SEM of three independent experiments, each conducted in triplicate. ( B ) siRNA-treated cells show a corresponding decrease in the protein expression of these genes.

Article Snippet: The mRNA target sequences for HSP27 (Gene ID: 3315), cFLIP (Gene ID: 8837), and CLU (Gene ID: 1191) were designed using a siRNA template design tool (Ambion, Austin, TX, USA).

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Medicines

Article Title: Triple Silencing of HSP27, cFLIP, and CLU Genes Promotes the Sensitivity of Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells

doi: 10.3390/medicines11030007

Figure Lengend Snippet: Effect of doxazosin on the viability of PC-3 cells subjected to single or dual siRNA silencing of HSP27, cFLIP, and CLU genes. PC-3 cells were treated with 0, 10, or 25 µM doxazosin for 24 h, and then cell viability was measured by the MTT assay. ( A ) Cell viability was significantly decreased at 25 µM doxazosin in cells with CLU gene silencing, while the silencing of HSP27 and cFLIP genes did not show this effect. ( B ) Dual siRNA silencing (HSP27 and cFLIP, HSP27 and CLU, or cFLIP and CLU) resulted in a significant decrease in cell viability at both 10 and 25 µM doxazosin. Data represent the mean ± SEM of three independent experiments, each conducted in triplicate. Statistical significance ( p < 0.05) was determined by a one-way ANOVA and Tukey’s test followed by a Student’s t -test for 0 µM vs. 1, 10, and 25 µM comparisons.

Article Snippet: The mRNA target sequences for HSP27 (Gene ID: 3315), cFLIP (Gene ID: 8837), and CLU (Gene ID: 1191) were designed using a siRNA template design tool (Ambion, Austin, TX, USA).

Techniques: MTT Assay

Journal: Medicines

Article Title: Triple Silencing of HSP27, cFLIP, and CLU Genes Promotes the Sensitivity of Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells

doi: 10.3390/medicines11030007

Figure Lengend Snippet: Effect of doxazosin on the viability of PC-3 cells subjected to triple siRNA silencing of HSP27, cFLIP, and CLU genes. Cell viability was significantly decreased at 10 and 25 μM doxazosin. In addition, even at 1 μM doxazosin, cell viability was significantly reduced compared to cells subjected to dual siRNA targeting two genes. Data represent the mean ± SEM of three independent experiments, each conducted in triplicate. Statistical significance ( p < 0.05) was determined by a one-way ANOVA and Tukey’s test followed by a Student’s t -test for 0 µM vs. 1, 10, and 25 µM comparisons.

Article Snippet: The mRNA target sequences for HSP27 (Gene ID: 3315), cFLIP (Gene ID: 8837), and CLU (Gene ID: 1191) were designed using a siRNA template design tool (Ambion, Austin, TX, USA).

Techniques: