Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene
CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrati...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-06-01
|
| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050123000207 |
| _version_ | 1797807212810207232 |
|---|---|
| author | Nanna S. Mikkelsen Sabina S. Hernandez Trine I. Jensen Jessica L. Schneller Rasmus O. Bak |
| author_facet | Nanna S. Mikkelsen Sabina S. Hernandez Trine I. Jensen Jessica L. Schneller Rasmus O. Bak |
| author_sort | Nanna S. Mikkelsen |
| collection | DOAJ |
| description | CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies. |
| first_indexed | 2024-03-13T06:19:11Z |
| format | Article |
| id | doaj.art-f2b78d2a32624f20b177aadcf3362a66 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-03-13T06:19:11Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-f2b78d2a32624f20b177aadcf3362a662023-06-10T04:27:40ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012023-06-0129116Enrichment of transgene integrations by transient CRISPR activation of a silent reporter geneNanna S. Mikkelsen0Sabina S. Hernandez1Trine I. Jensen2Jessica L. Schneller3Rasmus O. Bak4Department of Biomedicine, Aarhus University, Aarhus C, DenmarkDepartment of Biomedicine, Aarhus University, Aarhus C, DenmarkDepartment of Biomedicine, Aarhus University, Aarhus C, DenmarkDepartment of Biomedicine, Aarhus University, Aarhus C, Denmark; RNA and Gene Therapies, Novo Nordisk A/S, Maaloev, DenmarkDepartment of Biomedicine, Aarhus University, Aarhus C, Denmark; Aarhus Institute of Advanced Studies, Aarhus University, Aarhus C, Denmark; Corresponding author: Rasmus O. Bak, Aarhus University, Department of Biomedicine, Høegh-Guldbergsgade 10, Bldg. 1115, 8000 Aarhus C, Denmark.CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.http://www.sciencedirect.com/science/article/pii/S2329050123000207transgene integrationenrichmentselectionCRISPRreporter geneCAR T |
| spellingShingle | Nanna S. Mikkelsen Sabina S. Hernandez Trine I. Jensen Jessica L. Schneller Rasmus O. Bak Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene Molecular Therapy: Methods & Clinical Development transgene integration enrichment selection CRISPR reporter gene CAR T |
| title | Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene |
| title_full | Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene |
| title_fullStr | Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene |
| title_full_unstemmed | Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene |
| title_short | Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene |
| title_sort | enrichment of transgene integrations by transient crispr activation of a silent reporter gene |
| topic | transgene integration enrichment selection CRISPR reporter gene CAR T |
| url | http://www.sciencedirect.com/science/article/pii/S2329050123000207 |
| work_keys_str_mv | AT nannasmikkelsen enrichmentoftransgeneintegrationsbytransientcrispractivationofasilentreportergene AT sabinashernandez enrichmentoftransgeneintegrationsbytransientcrispractivationofasilentreportergene AT trineijensen enrichmentoftransgeneintegrationsbytransientcrispractivationofasilentreportergene AT jessicalschneller enrichmentoftransgeneintegrationsbytransientcrispractivationofasilentreportergene AT rasmusobak enrichmentoftransgeneintegrationsbytransientcrispractivationofasilentreportergene |