Schjeide Brit-Maren Michaud, Schenke Maren, Seeger Bettina, Püschel Gerhard Paul
Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam, 14558 Nuthetal, Germany.
Department of Nutritional Biochemistry, Faculty of Life Sciences: Food, Nutrition and Health, University of Bayreuth, 95326 Kulmbach, Germany.
Methods Protoc. 2022 May 25;5(3):43. doi: 10.3390/mps5030043.
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 (, , , , , and ) 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.
为了改进最近建立的用于评估肉毒杆菌神经毒素效力的基于细胞的检测方法，利用CRISPR/Cas9技术，对源自神经母细胞瘤的SiMa细胞和诱导多能干细胞（iPSC）进行改造，将报告荧光素酶的编码序列整合到基因安全位点。开发了一种新方法——双对照定量拷贝数PCR（dc-qcnPCR），用于检测供体DNA的脱靶整合。分析了每种细胞类型克隆中的供体DNA插入成功率和靶向插入成功率。dc-qcnPCR能够可靠地定量两种细胞系中的拷贝数。与iPSC相比，SiMa细胞中供体DNA错误整合的概率显著增加。这可能是因为SiMa克隆中一些双链修复基因（、、、、、和）的相对基因表达量比iPSC克隆中的低。dc-qcnPCR提供了一种高效且经济的方法来检测CRISPR/Cas9诱导的供体DNA脱靶整合。
