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