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.…
| Author details: | Brit-Maren SchjeideORCiDGND, Maren SchenkeORCiDGND, Bettina SeegerORCiD, Gerhard PüschelORCiDGND |
|---|---|
| DOI: | https://doi.org/10.3390/mps5030043 |
| ISSN: | 2409-9279 |
| Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/35736544 |
| Title of parent work (English): | Methods and protocols : M&Ps |
| Publisher: | MDPI |
| Place of publishing: | Basel, Schweiz |
| Further contributing person(s): | Antónia Monteiro |
| Publication type: | Article |
| Language: | English |
| Date of first publication: | 2022/05/25 |
| Publication year: | 2022 |
| Release date: | 2022/09/27 |
| Tag: | CRISPR editing validation; copy number analyses; homologous recombination deficiency; homology-directed repair |
| Volume: | 5 |
| Issue: | 3 |
| Article number: | 43 |
| Number of pages: | 14 |
| First page: | 1 |
| Last Page: | 14 |
| Funding institution: | German Federal Ministry of Education and Research |
| Funding institution: | Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) |
| Funding number: | 031L0132A/B |
| Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Ernährungswissenschaft |
| Extern / Extern | |
| DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
| 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit | |
| Peer review: | Referiert |
| Grantor: | Publikationsfonds der Universität Potsdam |
| Publishing method: | Open Access / Gold Open-Access |
| DOAJ gelistet | |
| License (German): |  CC-BY - Namensnennung 4.0 International |
| External remark: | Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 1269 |
