Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification.
Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification.
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一种新的双控定量拷贝数聚合酶链式反应方法用于定量CRISPR诱导的DNA修饰后的非靶向转基因整合。
DOI：
10.3390/mps5030043
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发表时间：
2022-05-25
影响因子：
2.4
通讯作者：
Puschel, Gerhard Paul
中科院分区：
文献类型：
--
作者：
Schjeide, Brit-Maren Michaud;Schenke, Maren;Seeger, Bettina;Puschel, Gerhard Paul
为了改进最近建立的基于细胞的测定以评估肉毒杆菌神经毒素的效力，对神经母细胞瘤衍生的SiMa细胞和诱导的多能干细胞（iPSC）进行修饰，以利用CRISPR/Cas9将报告荧光素酶的编码序列掺入遗传安全港中。建立了一种检测供体DNA脱靶整合的新方法--双控定量拷贝数PCR（dc-qcnPCR）。在每种细胞类型的克隆中分析供体DNA插入成功率和靶向插入成功率。dc-qcnPCR可靠地定量了两种细胞系中的拷贝数。与iPSC相比，SiMa细胞中不正确的供体DNA整合的可能性显著增加。这可能是由于SiMa克隆中许多双链修复基因（BRCA 1、DNA 2、EXO 1、MCPH 1、MRE 11和RAD 51）的捆绑相对基因表达低于iPSC克隆。dc-qcnPCR提供了一种有效且具有成本效益的方法来检测脱靶CRISPR/Cas9诱导的供体DNA整合。
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-target CRISPR/Cas9-induced donor DNA integrations.
DOI：
10.1258/ebm.2011.011107
发表时间：
2011-09
期刊：
Experimental biology and medicine (Maywood, N.J.)
影响因子：
--
作者：
Tichy ED
通讯作者：
Tichy ED