Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification
- In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-targetIn order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations.…
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| Author details: | Brit-Maren Michaud SchjeideORCiDGND, Maren SchenkeORCiDGND, Bettina SeegerORCiD, Gerhard Paul PüschelORCiDGND |
|---|---|
| URN: | urn:nbn:de:kobv:517-opus4-561755 |
| DOI: | https://doi.org/10.25932/publishup-56175 |
| ISSN: | 1866-8372 |
| Title of parent work (German): | Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe |
| Publication series (Volume number): | Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe (1269) |
| Publisher: | Universitätsverlag Potsdam |
| Place of publishing: | Potsdam |
| Further contributing person(s): | Antónia Monteiro |
| Publication type: | Postprint |
| Language: | English |
| Date of first publication: | 2022/09/27 |
| Publication year: | 2022 |
| Publishing institution: | Universität Potsdam |
| Release date: | 2022/09/27 |
| Tag: | CRISPR editing validation; copy number analyses; homologous recombination deficiency; homology-directed repair |
| Number of pages: | 14 |
| First page: | 1 |
| Last Page: | 14 |
| Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Ernährungswissenschaft |
| DDC classification: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
| Peer review: | Referiert |
| Publishing method: | Open Access / Green Open-Access |
| License (German): |  CC-BY - Namensnennung 4.0 International |
| External remark: | Bibliographieeintrag der Originalveröffentlichung/Quelle |
