Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing.

Journal Article (Journal Article)

Genetic manipulation via transgene overexpression, RNAi, or Cas9-based methods is central to biomedical research. Unfortunately, use of these tools is often limited by vector options. We have created a modular platform (pMVP) that allows a gene of interest to be studied in the context of an array of promoters, epitope tags, conditional expression modalities, and fluorescent reporters, packaged in 35 custom destination vectors, including adenovirus, lentivirus, PiggyBac transposon, and Sleeping Beauty transposon, in aggregate >108,000 vector permutations. We also used pMVP to build an epigenetic engineering platform, pMAGIC, that packages multiple gRNAs and either Sa-dCas9 or x-dCas9(3.7) fused to one of five epigenetic modifiers. Importantly, via its compatibility with adenoviral vectors, pMAGIC uniquely enables use of dCas9/LSD1 fusions to interrogate enhancers within primary cells. To demonstrate this, we used pMAGIC to target Sa-dCas9/LSD1 and modify the epigenetic status of a conserved enhancer, resulting in altered expression of the homeobox transcription factor PDX1 and its target genes in pancreatic islets and insulinoma cells. In sum, the pMVP and pMAGIC systems empower researchers to rapidly generate purpose-built, customized vectors for manipulation of gene expression, including via targeted epigenetic modification of regulatory elements in a broad range of disease-relevant cell types.

Full Text

Duke Authors

Cited Authors

  • Haldeman, JM; Conway, AE; Arlotto, ME; Slentz, DH; Muoio, DM; Becker, TC; Newgard, CB

Published Date

  • February 28, 2019

Published In

Volume / Issue

  • 47 / 4

Start / End Page

  • e23 -

PubMed ID

  • 30590691

Pubmed Central ID

  • PMC6393299

Electronic International Standard Serial Number (EISSN)

  • 1362-4962

Digital Object Identifier (DOI)

  • 10.1093/nar/gky1286


  • eng

Conference Location

  • England