{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,29]],"date-time":"2026-03-29T13:01:18Z","timestamp":1774789278526,"version":"3.50.1"},"reference-count":33,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2018,3,22]],"date-time":"2018-03-22T00:00:00Z","timestamp":1521676800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/ijms19040946","type":"journal-article","created":{"date-parts":[[2018,3,22]],"date-time":"2018-03-22T14:39:31Z","timestamp":1521729571000},"page":"946","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["CRISPR\/Cas9-Based Cellular Engineering for Targeted Gene Overexpression"],"prefix":"10.3390","volume":"19","author":[{"given":"Mark","family":"Osborn","sequence":"first","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Institute for Engineering in Medicine, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Asan-Minnesota Institute for Innovating Transplantation, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christopher","family":"Lees","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4169-0015","authenticated-orcid":false,"given":"Amber","family":"McElroy","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5067-1281","authenticated-orcid":false,"given":"Sarah","family":"Merkel","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7314-2550","authenticated-orcid":false,"given":"Cindy","family":"Eide","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6183-2132","authenticated-orcid":false,"given":"Wendy","family":"Mathews","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Colby","family":"Feser","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Madison","family":"Tschann","sequence":"additional","affiliation":[{"name":"Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ron","family":"McElmury","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3950-8747","authenticated-orcid":false,"given":"Beau","family":"Webber","sequence":"additional","affiliation":[{"name":"Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Department of Pediatrics, Division of Hematology, Oncology, and Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chong","family":"Kim","sequence":"additional","affiliation":[{"name":"Asan-Minnesota Institute for Innovating Transplantation, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Asan Institute for Life Sciences, Asan Medical Center, Seoul 138-736, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruce","family":"Blazar","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jakub","family":"Tolar","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Blood and Marrow Transplantation, Medical School, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Asan-Minnesota Institute for Innovating Transplantation, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2018,3,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1006\/geno.1994.1239","article-title":"Structural organization of the human type vii collagen gene (COL7A1), composed of more exons than any previously characterized gene","volume":"21","author":"Christiano","year":"1994","journal-title":"Genomics"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"20256","DOI":"10.1016\/S0021-9258(17)31984-1","article-title":"Cloning of human type VII collagen. Complete primary sequence of the alpha 1(VII) chain and identification of intragenic polymorphisms","volume":"269","author":"Christiano","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.clindermatol.2004.09.015","article-title":"Progress in epidermolysis bullosa: From eponyms to molecular genetic classification","volume":"23","author":"Uitto","year":"2005","journal-title":"Clin. Dermatol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1038\/nm1063","article-title":"Injection of recombinant human type vii collagen restores collagen function in dystrophic epidermolysis bullosa","volume":"10","author":"Woodley","year":"2004","journal-title":"Nat. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1172\/JCI200317193","article-title":"Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue","volume":"111","author":"Lin","year":"2003","journal-title":"J. Clin. Investig."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1025","DOI":"10.1111\/bjd.12599","article-title":"Fibroblast cell therapy enhances initial healing in recessive dystrophic epidermolysis bullosa wounds: Results of a randomized, vehicle-controlled trial","volume":"169","author":"Petrof","year":"2013","journal-title":"Br. J. Dermatol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1346","DOI":"10.1016\/j.jid.2016.02.811","article-title":"Gene-corrected fibroblast therapy for recessive dystrophic epidermolysis bullosa using a self-inactivating COL7A1 retroviral vector","volume":"136","author":"Jackow","year":"2016","journal-title":"J. Investig. Dermatol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1151","DOI":"10.1038\/mt.2013.56","article-title":"Talen-based gene correction for epidermolysis bullosa","volume":"21","author":"Osborn","year":"2013","journal-title":"Mol. Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1038\/ng1041","article-title":"Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa","volume":"32","author":"Chen","year":"2002","journal-title":"Nat. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1046\/j.1523-1747.2003.12571.x","article-title":"Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type vii collagen at the basement membrane zone","volume":"121","author":"Woodley","year":"2003","journal-title":"J. Investig. Dermatol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1509","DOI":"10.1038\/mt.2010.91","article-title":"Sin retroviral vectors expressing COL7A1 under human promoters for ex vivo gene therapy of recessive dystrophic epidermolysis bullosa","volume":"18","author":"Titeux","year":"2010","journal-title":"Mol. Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1038\/JID.2015.364","article-title":"Lentiviral engineered fibroblasts expressing codon-optimized COL7A1 restore anchoring fibrils in RDEB","volume":"136","author":"Georgiadis","year":"2016","journal-title":"J. Investig. Dermatol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1166","DOI":"10.1038\/nm766","article-title":"Stable nonviral genetic correction of inherited human skin disease","volume":"8","author":"Thyagarajan","year":"2002","journal-title":"Nat. Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"836","DOI":"10.1016\/j.jid.2016.11.038","article-title":"Correction of recessive dystrophic epidermolysis bullosa by transposon-mediated integration of COL7A1 in transplantable patient-derived primary keratinocytes","volume":"137","author":"Latella","year":"2017","journal-title":"J. Investig. Dermatol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1770","DOI":"10.1182\/blood-2007-01-068759","article-title":"Hot spots of retroviral integration in human CD34+ hematopoietic cells","volume":"110","author":"Cattoglio","year":"2007","journal-title":"Blood"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"964","DOI":"10.1172\/JCI37630","article-title":"The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy","volume":"119","author":"Montini","year":"2009","journal-title":"J. Clin. Investig."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1016\/j.bbrc.2016.06.094","article-title":"Detection of sleeping beauty transposition in the genome of host cells by non-radioactive southern blot analysis","volume":"477","author":"Aravalli","year":"2016","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Webber, B.R., Osborn, M.J., McElroy, A.N., Twaroski, K., Lonetree, C.L., DeFeo, A.P., Xia, L., Eide, C., Lees, C.J., and McElmurry, R.T. (2016). Crispr\/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa. NPJ Regen. Med., 1.","DOI":"10.1038\/npjregenmed.2016.14"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2591","DOI":"10.1093\/nar\/gkt1224","article-title":"Megatals: A rare-cleaving nuclease architecture for therapeutic genome engineering","volume":"42","author":"Boissel","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1200","DOI":"10.1038\/mt.2008.114","article-title":"Zinc-finger nucleases: The next generation emerges","volume":"16","author":"Cathomen","year":"2008","journal-title":"Mol. Ther."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1016\/j.scr.2014.02.004","article-title":"Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells","volume":"12","author":"Tiyaboonchai","year":"2014","journal-title":"Stem Cell Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"634","DOI":"10.3324\/haematol.2015.138594","article-title":"Reduced intensity haplo plus single cord transplant compared to double cord transplant: Improved engraftment and graft-versus-host disease-free, relapse-free survival","volume":"101","author":"Hari","year":"2016","journal-title":"Haematologica"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1053\/j.seminhematol.2016.07.004","article-title":"Haploidentical cord transplantation-the best of both worlds","volume":"53","author":"Childs","year":"2016","journal-title":"Semin. Hematol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Hsiau, T., Maures, T., Waite, K., Yang, J., Kelso, R., Holden, K., and Stoner, R. (2018). Inference of crispr edits from sanger trace data. BioRxiv.","DOI":"10.1101\/251082"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/nature14136","article-title":"Genome-scale transcriptional activation by an engineered crispr-CAS9 complex","volume":"517","author":"Konermann","year":"2015","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"851","DOI":"10.1038\/nbt.1562","article-title":"Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases","volume":"27","author":"Hockemeyer","year":"2009","journal-title":"Nat. Biotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1038\/nbt.3290","article-title":"Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells","volume":"33","author":"Hendel","year":"2015","journal-title":"Nat. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1640","DOI":"10.1038\/mt.2010.132","article-title":"A ubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors","volume":"18","author":"Zhang","year":"2010","journal-title":"Mol. Ther."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"4395","DOI":"10.1182\/blood-2011-03-340711","article-title":"Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine wiskott-aldrich syndrome","volume":"119","author":"Astrakhan","year":"2012","journal-title":"Blood"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1577","DOI":"10.1093\/nar\/gkv019","article-title":"A minimal ubiquitous chromatin opening element (UCOE) effectively prevents silencing of juxtaposed heterologous promoters by epigenetic remodeling in multipotent and pluripotent stem cells","volume":"43","author":"Ackermann","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2118","DOI":"10.1074\/jbc.M108779200","article-title":"The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa","volume":"277","author":"Chen","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1256","DOI":"10.1038\/nbt.3408","article-title":"Homology-driven genome editing in hematopoietic stem and progenitor cells using zfn mrna and AAV6 donors","volume":"33","author":"Wang","year":"2015","journal-title":"Nat. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Qasim, W., Zhan, H., Samarasinghe, S., Adams, S., Amrolia, P., Stafford, S., Butler, K., Rivat, C., Wright, G., and Somana, K. (2017). Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci. Transl. Med., 9.","DOI":"10.1126\/scitranslmed.aaj2013"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/4\/946\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T14:58:06Z","timestamp":1760194686000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/4\/946"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,3,22]]},"references-count":33,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2018,4]]}},"alternative-id":["ijms19040946"],"URL":"https:\/\/doi.org\/10.3390\/ijms19040946","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,3,22]]}}}