Crispr/cas9-based cellular engineering for targeted gene overexpression

Mark J. Osborn, Christopher J. Lees, Amber N. McElroy, Sarah C. Merkel, Cindy R. Eide, Wendy Mathews, Colby J. Feser, Madison Tschann, Ron T. McElmury, Beau R. Webber, Chong Jai Kim, Bruce R. Blazar, Jakub Tolar

Research output: Contribution to journalArticlepeer-review

16 Scopus citations


Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

Original languageEnglish (US)
Article number946
JournalInternational journal of molecular sciences
Issue number4
StatePublished - Apr 2018

Bibliographical note

Funding Information:
Acknowledgments: We appreciate funding support from The Children’s Cancer Research Fund, The Kidz First Fund & the Corrigan Family. Jakub Tolar is supported in part by R01 AR063070 and P01 CA065493. Mark J. Osborn is supported by a grant from Regenerative Medicine of Minnesota.

Publisher Copyright:
© 2018 by the authors. Licensee MDPI, Basel, Switzerland.


  • Adeno-associated virus
  • CRISPR/Cas9
  • Cord blood
  • Homology directed repair
  • Recessive dystrophic epidermolysis bullosa
  • T-cells
  • Transcriptional activation
  • Ubiquitous chromatin opening element


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