{"title":"在中国仓鼠卵巢细胞 18S rDNA 基因座上精确整合抗 CD52 双单克隆抗体表达构建体时,对成对 Cas9 快速酶和 RNA 引导的 FokI 基因组编辑工具的评估。","authors":"Hadi Bayat , Faranak Farahmand , Sayed Hassan Tabatabaee , Forough Shams , Omid Mohammadian , Es'hagh Pourmaleki , Azam Rahimpour","doi":"10.1016/j.pep.2024.106445","DOIUrl":null,"url":null,"abstract":"<div><h3>Introduction</h3><p>The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus.</p></div><div><h3>Methods</h3><p>T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5′- and 3′-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays.</p></div><div><h3>Results</h3><p>Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells.</p></div><div><h3>Conclusion</h3><p>Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4000,"publicationDate":"2024-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus\",\"authors\":\"Hadi Bayat , Faranak Farahmand , Sayed Hassan Tabatabaee , Forough Shams , Omid Mohammadian , Es'hagh Pourmaleki , Azam Rahimpour\",\"doi\":\"10.1016/j.pep.2024.106445\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Introduction</h3><p>The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus.</p></div><div><h3>Methods</h3><p>T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5′- and 3′-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays.</p></div><div><h3>Results</h3><p>Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells.</p></div><div><h3>Conclusion</h3><p>Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. 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引用次数: 0
摘要
导言:本研究的目的是比较两种基于CRISPR/Cas9的正交策略--成对Cas9缺口酶(paired-Cas9n)和RNA引导的FokI(RFN)--在中国仓鼠卵巢(CHO)细胞中靶向18S rDNA位点并将抗CD52单克隆抗体(mAb)双链表达盒精确整合到该位点的能力:方法:使用T7E1和高分辨率熔融(HRM)测定法比较上述系统在靶位点诱导双链断裂(DSB)的能力。此外,还使用 5'- 和 3'- 连接聚合酶链反应(PCR)来验证 mAb 表达盒与 18S rDNA 基因座靶向整合的准确性。最后,测量了抗 CD52 mAb 基因的拷贝数,并使用 ELISA 和 Western-blotting 方法分析了其表达情况:结果:我们的研究结果表明,配对-Cas9n 和 RFN 都能在靶位点诱导 DSB,但 RFN 在 HRM 分析中的表现稍好一些。我们还证实抗 CD52 mAb 基因盒准确整合到了 18S rDNA 基因座上,并且 mAb 在 CHO 细胞中成功表达:综上所述,我们的研究结果阐明了成对Cas9n和RFN基因组编辑工具在靶向18S rDNA位点方面的前景。利用交界 PCR 技术表明,使用 RFN 构建的 mAb 表达盒可与 CHO-K1 细胞基因组进行定点整合。此外,利用基于双螺旋内部核糖体进入位点(IRES)的载体系统,抗 CD52 mAb 可以在 18S rDNA 目标位点正确表达。
Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus
Introduction
The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus.
Methods
T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5′- and 3′-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays.
Results
Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells.
Conclusion
Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.
期刊介绍:
Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.