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Open AccessArticle

CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression

1
Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA
2
Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
3
Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
4
Institute for Engineering in Medicine, University of Minnesota, Minneapolis, MN 55455, USA
5
Asan-Minnesota Institute for Innovating Transplantation, University of Minnesota, Minneapolis, MN 55455, USA
6
Department of Pediatrics, Division of Hematology, Oncology, and Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA
7
Asan Institute for Life Sciences, Asan Medical Center, Seoul 138-736, Korea
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(4), 946; https://doi.org/10.3390/ijms19040946
Received: 23 February 2018 / Revised: 15 March 2018 / Accepted: 18 March 2018 / Published: 22 March 2018
(This article belongs to the Special Issue Genome Editing 2018)
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. View Full-Text
Keywords: CRISPR/Cas9; recessive dystrophic epidermolysis bullosa; transcriptional activation; homology directed repair; adeno-associated virus; ubiquitous chromatin opening element; T-cells; cord blood CRISPR/Cas9; recessive dystrophic epidermolysis bullosa; transcriptional activation; homology directed repair; adeno-associated virus; ubiquitous chromatin opening element; T-cells; cord blood
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Osborn, M.J.; Lees, C.J.; McElroy, A.N.; Merkel, S.C.; Eide, C.R.; Mathews, W.; Feser, C.J.; Tschann, M.; McElmury, R.T.; Webber, B.R.; Kim, C.J.; Blazar, B.R.; Tolar, J. CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression. Int. J. Mol. Sci. 2018, 19, 946.

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