O⁶-甲基鸟嘌呤-DNA甲基转移酶（O⁶-Methylguanine-DNA methyltransferase，MGMT）基因是一个重要的DNA甲基化标志物，美国国立综合癌症网络（National Comprehensive Cancer Network, NCCN）中枢神经系统肿瘤临床实践指南（2020. v3版）中明确指出胶质母细胞瘤（glioblastoma, GBM）患者基于年龄、卡氏评分基础之上应进行MGMT基因启动子甲基化检测，以评估对化疗药物替莫唑胺（temozolomide，TMZ）的反应。作为临床实验室，为了得到准确可靠且互通性良好的检测结果，需要合适的甲基化检测质控品。国际人类表观基因组联盟（International Human Epigenome Consortium，IHEC）指出，构建异质性甲基化模式的甲基化检测质控品是确保临床实验室检测结果准确性和标准化的重要保证。因此，对于MGMT基因甲基化检测需要制备新型质控品，既要满足异质性的甲基化模式，同时甲基化修饰应当覆盖启动子区域关键调控CpG位点，以具备模拟肿瘤患者体内MGMT甲基化状态的性能特点。

    本研究基于新兴的CRISPR/Cas9表观遗传编辑技术，利用CRISPR/Cas9介导的DNA双链断裂（double strand breaks，DSB）和同源重组（homology-directed repair，HDR）的方式在HeLa细胞内靶向MGMT基因启动子甲基化，通过构建MGMT基因甲基化的单克隆细胞系用于制备新型甲基化检测质控品。为了最终成功筛选得到MGMT基因甲基化的单克隆细胞系，本研究需要解决的核心问题包括两方面：一方面，探究CRISPR/Cas9表观遗传编辑工具在HeLa 细胞靶向MGMT甲基化的有效性和稳定性；另一方面，提高CRISPR/Cas9表观遗传基因编辑效率，克服大分子量CRISPR质粒介导的HDR效率低下的技术局限性。

    研究内容分成以下两部分：（1）利用设计合成的不同单链DNA（single-stranded DNA，ssDNA）模板验证CRISPR/Cas9介导的DSB通过DNA损伤修复通路在HeLa细胞内靶向MGMT基因启动子甲基化的效率和稳定性。同时，探究靶向甲基化对MGMT 和DNA甲基转移酶（DNA methyltransferases，DNMTs）mRNA 表达水平的调控作用。（2）在ssDNA模板基础上进一步设计双链DNA构建的质粒（dsDNA-pUC57）通过M.SssI修饰作为长链甲基化同源模板，探究DNA连接酶IV抑制剂SCR7对长链甲基化模板介导的表观遗传重组效率的作用。依据优化的条件，一方面，在混合克隆和筛选的单克隆系分别验证靶向甲基化水平的稳定性。另一方面，检测MGMT 和DNMTs mRNA 表达水平，并进一步对靶向甲基化介导的体内外MGMT蛋白表达水平进行验证。

    结果显示：（1）利用外源性合成的甲基化ssDNA 同源模板，CRISPR/Cas9介导的DSB 通过HDR通路在HeLa细胞能够成功介导MGMT基因启动子发生甲基化改变，同时抑制MGMT 基因转录和上调DNMT1、UHRF1 mRNA表达水平。但是，ssDNA靶向甲基化水平随细胞传代数日后逐渐消失。（2）相比于ssDNA，长链甲基化同源模板介导的HDR效率显著下降。通过SCR7给药能够以浓度依赖方式提高长链甲基化同源模板介导的表观遗传重组效率。经流式分选细胞传代培养40天后，混合克隆和筛选的HDR阳性单克隆细胞系通过不同甲基化定量检测方法证实靶向介导的MGMT基因高甲基化改变不会随细胞传代消失，伴随细胞分化甲基化水平能够稳定表达。并且，靶向MGMT基因甲基化能够有效抑制转录。相比于ssDNA，长链甲基化同源模板能够介导更多DNMTs的表达。在蛋白水平，构建的MGMT基因甲基化单克隆细胞系通过体内外实验证实蛋白表达完全缺失。因此，SCR7联合CRIPSR/Cas9、长链甲基化dsDNA同源模板作为一种优化的方法能够有效介导MGMT基因启动子甲基化并造成基因沉默。

    综上所述，本研究的创新性在于采用一种优化的CRISPR/Cas9表观遗传编辑方法提高了外源性长链甲基化同源模板介导的表观遗传重组效率，同时有效介导了基因编辑的HeLa细胞内MGMT甲基化水平的稳定表达。本研究成功构建一株新型MGMT基因甲基化的单克隆细胞系，该细胞系具有异质性甲基化模式，适宜作为甲基化检测质控品。在今后工作中，进一步将构建的MGMT基因甲基化的单克隆细胞系制备成满足临床实验室检测需要的新型甲基化检测质控品用于开展室间质评活动，以完善此部分研究内容。另外，本研究所采用的优化的CRISPR/Cas9靶向甲基化技术通过表观遗传沉默方式造成基因沉默，对于特定基因表观遗传机制研究、甲基化细胞及动物模型构建、肿瘤基因治疗的应用提供了新的思路和方法。

    O⁶-methylguanine-DNA methyltransferase (MGMT) is a critical DNA methylation biomarker. According to National Comprehensive Cancer Network (NCCN) Guideline for Central Nervous System Cancer (Version 3. 2021), GBM patients need to perform MGMT methylation detection based on age and Karnofsky score, so as to assess the response to temozolomide (TMZ). For the clinical laboratories, in order to enhance accuracy and reliability, a proper quality control material for DNA methylation detection is extremely needed. The International Human Epigenome Consortium (IHEC) indicated that the preparation of quality control material with heterogeneous methylation pattern for methylation testing is important for the accuracy and standardization of results among clinical laboratories. Therefore, the preparation of novel quality control material for MGMT methylation detection not only need to have heterogeneous methylation pattern, but also possess hypermethylation in gene promoter critical CpG sites, thus mimicking the real DNA methylation profiles in tumor patients.

    Based on CRISPR/Cas9 epigenetic editing tool, our study utilized CRISPR/Cas9-directed double strand breaks (DSB) followed by homology-directed repair (HDR) pathway to target MGMT promoter methylation alterations, and then generated MGMT methylated monoclonal cell line used for the preparation of novel quality control material for methylation testing. In order to successfully select MGMT methylated monoclonal cell line using for the preparation of quality control material, the core problem in our study is the solution for the stability of targeted DNA methylation status and the poor HDR efficiency for CRISPR/Cas9.

    Our experiment was carried out in two parts as follows. i. We firstly designed different single-stranded DNA (ssDNA) templates to explore the targeted MGMT methylation efficiency and stability via CRISPR/Cas9-directed DSB followed by DNA damage repair pathway. Meanwhile, we also studied the regulation on MGMT and DNMTs mRNA expression caused by targeted MGMT methylation alterations. ii. According to ssDNA templates, we subsequently designed dsDNA repair template (dsDNA-pUC57 plasmid) with M.SssI modification as long-methylated repair template. Based on SCR7 treatment, we studied the role of SCR7 on dsDNA-mediated epigenetic recombination efficiency. On the basis of optimized method, we verified the steady of targeted methylation status for both mixed clones and monoclonal cell lines after culturing for 40 days by flow cytometry. In addition, we also explored the mRNA expression of MGMT and DNMTs, and further detected the expression level of MGMT protein induced by targeting methylation both in vitro and in vivo.

    The results showed：i. CRISPR/Cas9-directed DSB following HDR damage repair pathway can successfully induce MGMT gene methylation alteration, inhibit MGMT mRNA expression, as well as enhance DNMT1 and UHRF1expression level. However, ssDNA repair template had limited ability to generate efficient methylation level, since the targeted methylation status dropped rapidly after cell passage for several days. ii. Compared to ssDNA, the long-methylated dsDNA repair template-directed HDR efficiency reduced obviously. SCR7 treatment can increase the epigenetic recombination rate guided by long-methylated dsDNA in a concentration dependent manner. The sorted cells by flow cytometry had been confirmed with stably hypermethylation alteration in MGMT promoter by several different DNA methylation quantitative detection methods after culturing for 40 days. Meanwhile, targeting MGMT methylation can suppress gene transcription and induce higher expression levels of DNMTs compared to ssDNA templates. Additionally, the establishment of MGMT methylation monoclonal cell line demonstrated complete loss of MGMT protein expression both in vitro and in vivo. Therefore, a combination of SCR7 treatment, CRISPR/Cas9 components, and long-methylated dsDNA templates as an optimized strategy can result in hypermethylation alteration of targeted MGMT gene and gene silencing, through the enhancement of epigenetic recombination efficiency.

    In conclusion, the novelty of our study lies in the utilization of an optimized method of CRISPR/Cas9 epigenetic editing tool to successfully improve the epigenetic recombination efficiency directed by exogenous long-methylated repair template. Meanwhile, our optimized method enables the stable status of targeted MGMT gene methylation in edited HeLa cells. Our research successfully generated a new MGMT methylation monoclonal cell line with heterogeneous methylation pattern, thus suitable as quality control material for MGMT methylation detection. In the future work, we will use this established MGMT methylation monoclonal cell line to produce novel quality control material for MGMT methylation detection, which meet clinical laboratory testing needs, and then implement external quality assessment activity. Furthermore, the optimized method used in our study can lead to gene silencing through epigenetic silencing manner, which provides new method and idea for the study of epigenetic mechanism toward specific genes，the establishment of methylated cell and animal models, as well as tumor gene therapy.