Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation
Genomic safe harbors (GSH) are defined as sites in the host genome that allow stable expression of inserted transgenes while having no adverse effects on the host cell, making them ideal for use in basic research and therapeutic applications. Silencing and fluctuations in transgene expression would...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-09-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S232905012200081X |
| _version_ | 1828315700000718848 |
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| author | Anne Inderbitzin Tom Loosli Roger D. Kouyos Karin J. Metzner |
| author_facet | Anne Inderbitzin Tom Loosli Roger D. Kouyos Karin J. Metzner |
| author_sort | Anne Inderbitzin |
| collection | DOAJ |
| description | Genomic safe harbors (GSH) are defined as sites in the host genome that allow stable expression of inserted transgenes while having no adverse effects on the host cell, making them ideal for use in basic research and therapeutic applications. Silencing and fluctuations in transgene expression would be highly undesirable effects. We have previously shown that transgene expression in Jurkat T cells is not silenced for up to 160 days after CRISPR-Cas9-mediated insertion of reporter genes into the adeno-associated virus site 1 (AAVS1), a commonly used GSH. Here, we studied fluctuations in transgene expression upon targeted insertion into the GSH AAVS1. We have developed an efficient method to generate and validate highly complex barcoded plasmid libraries to study transgene expression on the single-cell level. Its applicability is demonstrated by inserting the barcoded transgene Cerulean into the AAVS1 locus in Jurkat T cells via the CRISPR-Cas9 technology followed by next-generation sequencing of the transcribed barcodes. We observed large transcriptional variations over two logs for transgene expression in the GSH AAVS1. This barcoded transgene insertion model is a powerful tool to investigate fluctuations in transgene expression at any GSH site. |
| first_indexed | 2024-04-13T17:03:36Z |
| format | Article |
| id | doaj.art-ba4cc8c6271b472db29d5ef2822f7a11 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-04-13T17:03:36Z |
| publishDate | 2022-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-ba4cc8c6271b472db29d5ef2822f7a112022-12-22T02:38:33ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012022-09-0126107118Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variationAnne Inderbitzin0Tom Loosli1Roger D. Kouyos2Karin J. Metzner3Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland; Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Life Science Zurich Graduate School, University of Zurich, Zurich, SwitzerlandDepartment of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland; Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Life Science Zurich Graduate School, University of Zurich, Zurich, SwitzerlandDepartment of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland; Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, SwitzerlandDepartment of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland; Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Corresponding author Karin J. Metzner, MD, University Hospital Zurich, Department of Infectious Diseases and Hospital Epidemiology, Rämistrasse 100, 8091 Zurich, Switzerland.Genomic safe harbors (GSH) are defined as sites in the host genome that allow stable expression of inserted transgenes while having no adverse effects on the host cell, making them ideal for use in basic research and therapeutic applications. Silencing and fluctuations in transgene expression would be highly undesirable effects. We have previously shown that transgene expression in Jurkat T cells is not silenced for up to 160 days after CRISPR-Cas9-mediated insertion of reporter genes into the adeno-associated virus site 1 (AAVS1), a commonly used GSH. Here, we studied fluctuations in transgene expression upon targeted insertion into the GSH AAVS1. We have developed an efficient method to generate and validate highly complex barcoded plasmid libraries to study transgene expression on the single-cell level. Its applicability is demonstrated by inserting the barcoded transgene Cerulean into the AAVS1 locus in Jurkat T cells via the CRISPR-Cas9 technology followed by next-generation sequencing of the transcribed barcodes. We observed large transcriptional variations over two logs for transgene expression in the GSH AAVS1. This barcoded transgene insertion model is a powerful tool to investigate fluctuations in transgene expression at any GSH site.http://www.sciencedirect.com/science/article/pii/S232905012200081XCRISPR-Cas9barcoded transgenegenomic safe harborAAVS1Jurkat T cellsHIV-1 promoter |
| spellingShingle | Anne Inderbitzin Tom Loosli Roger D. Kouyos Karin J. Metzner Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation Molecular Therapy: Methods & Clinical Development CRISPR-Cas9 barcoded transgene genomic safe harbor AAVS1 Jurkat T cells HIV-1 promoter |
| title | Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation |
| title_full | Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation |
| title_fullStr | Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation |
| title_full_unstemmed | Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation |
| title_short | Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation |
| title_sort | quantification of transgene expression in gsh aavs1 with a novel crispr cas9 based approach reveals high transcriptional variation |
| topic | CRISPR-Cas9 barcoded transgene genomic safe harbor AAVS1 Jurkat T cells HIV-1 promoter |
| url | http://www.sciencedirect.com/science/article/pii/S232905012200081X |
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