CRISPR-Cas9系统在肿瘤生物学中的应用及前景

doi:10.12211/2096-8280.2022-054

合成生物学 ›› 2023, Vol. 4 ›› Issue (4): 703-719.DOI: 10.12211/2096-8280.2022-054

CRISPR-Cas9系统在肿瘤生物学中的应用及前景

马孟丹¹^,²^,³, 尚梦宇¹^,²^,⁴, 刘宇辰¹^,²

^1.深圳大学第一附属医院，深圳市第二人民医院，深圳转化医学研究院，广东深圳 518035
^2.广东省泌尿生殖肿瘤系统生物学与合成生物学重点实验室，广东深圳 518035
^3.汕头大学医学院，广东汕头 515041
^4.深圳大学医学部基础医学院，广东深圳 518060

收稿日期:2022-09-30 修回日期:2023-02-01 出版日期:2023-08-31 发布日期:2023-09-14
通讯作者: 刘宇辰
作者简介:马孟丹（1997—），女，硕士研究生。研究方向为医学合成生物学。E-mail：mamengdan7@163.com
刘宇辰（1988—），男，副研究员，研究生导师，博士后合作导师。研究方向为（1）肿瘤生物治疗和医学合成生物学；（2）创新肿瘤治疗新方法；（3）构建人工基因线路，肿瘤精准治疗。E-mail：liuyuchenmdcg@163.com
基金资助:
国家重点研发计划(2021YFA0911600)

Application and prospect of CRISPR-Cas9 system in tumor biology

MA Mengdan¹^,²^,³, SHANG Mengyu¹^,²^,⁴, LIU Yuchen¹^,²

^1.The first Affiliated Hospital of Shenzhen University，Shenzhen Second People′s Hospital，Shenzhen Institute of Translational Medicine，Shenzhen 518035，Guangdong，China
^2.Guangdong Provincial Key Laboratory of Systems Biology and Synthetic Biology for Urogenital Tumors，Shenzhen 518035，Guangdong，China
^3.Shantou University Medical College，Shantou 515041，Guangdong，China
^4.School of Basic Medical Sciences，Shenzhen University Health Science Center，Shenzhen 518060，China

Received:2022-09-30 Revised:2023-02-01 Online:2023-08-31 Published:2023-09-14
Contact: LIU Yuchen

摘要/Abstract

摘要：

RNA引导的CRISPR-Cas核酸酶系统最初作为适应性免疫系统的一部分在细菌中被发现，其修改或修饰遗传成分的能力已经带来了各种实际应用，如碱基编辑、转录调控和表观遗传修饰等。由于CRISPR-Cas基因编辑工具不仅功能强大，而且具有特异性强、效率高等特点，可以准确、快速地对整个基因组进行筛选，便于对特定疾病进行基因治疗，已被广泛应用于人类疾病治疗的相关研究。在肿瘤研究领域，CRISPR-Cas系统可以用来编辑基因组，探索肿瘤发生、发展和转移的机制。文章阐述了CRISPR-Cas9系统作为癌症研究工具所取得的进展；总结了该技术在癌症基础研究、诊断和治疗中的应用现状；讨论了这一技术在肿瘤研究新热点领域和临床医学精准医疗方面的发展前景，并指出了其面临的技术挑战和未来的发展方向。

Abstract:

The RNA-directed CRISPR-Cas nuclease system was first discovered in bacteria as part of the adaptive immune system, and its ability to modify genetic components has led to a variety of practical applications, such as base editing, insertion or deletion of long segments, transcriptional regulation, and epigenetic modification. Because CRISPR-Cas gene editing tool is not only powerful, but also highly specific and efficient, it can accurately and rapidly screen the whole genome and facilitate gene therapy for specific diseases, so that it has been widely used in related research on the treatment of human diseases. The occurrence of tumor is the result of malignant degeneration of normal cells caused by the combined action of multiple factors, multiple stages and multiple mechanisms. CRISPR-Cas gene editing technology can accurately change genetic information at the DNA level and simulate the corresponding malignant characteristics of cells caused by the change of genetic information, thus becoming a molecular mechanism to explore the occurrence, development and metastasis of tumors. To investigate signaling pathways related to drug resistance in tumors and develop potential approaches to gene and cell therapy for tumor therapy. From early acquisition of oncogene mutations to metastatic colonization of distant tissues and the development of drug resistance, this continuous process leaves clear phylogenetic signatures at each step. Mapping detailed cancer cell lineages using the CRISPR gene-editing tool can reveal the dynamic processes behind the development and progression of cancer metastases, which will help track tumor development patterns. Despite its rapid development, CRISPR-Cas system still has limitations in its delivery efficiency, safety, and off-target effects in tumor therapy. The progress of CRISPR-Cas9 as a cancer research tool is reviewed. The research progress of this technique in establishing tumor model, studying the mechanism of tumor development, diagnosis, treatment and lineage tracking of tumor development was summarized. The development prospects of this technology in the new hot areas of cancer research and precision medicine in clinical medicine were discussed, and the technical challenges and future development directions were pointed out.

Key words: CRISPR-associated protein, CRISPR-Cas derivatives, lineage tracking, cancer diagnosis, tumor therapy, screening drug targets

中图分类号:

Q819

马孟丹, 尚梦宇, 刘宇辰. CRISPR-Cas9系统在肿瘤生物学中的应用及前景[J]. 合成生物学, 2023, 4(4): 703-719.

MA Mengdan, SHANG Mengyu, LIU Yuchen. Application and prospect of CRISPR-Cas9 system in tumor biology[J]. Synthetic Biology Journal, 2023, 4(4): 703-719.

图/表 3

图1 CRISPR-Cas系统衍生工具的应用

Fig. 1 Application of CRISPR-Cas system derivatives

图2 与门遗传电路的设计与构建［50］（a）hUPⅡ和hTERT启动子是线路的输入端。hUPⅡ启动子驱动Cas9 mRNA的转录，hTERT启动子连接靶向LacI的sgRNA的转录。输出基因由LacI控制的CMV启动子驱动。效应子只有在Cas9蛋白和sgRNA同时存在时才能表达。（b）～（e）是合成线路（SC-1，SC-2，SC-3和SC-4）的示意图。输出基因分别为hRluc、hBAX、p21和E-cadherin

Fig. 2 Design and construction of the AND gate genetic circuits[50](a) The hUPⅡ and hTERT promoters are the inputs to the circuit. The hUPⅡ promoter drives the transcription of Cas9 mRNA and the hTERT promoter is linked to the transcription of sgRNA targeting LacI. The output gene is driven by a LacI-controlled CMV promoter. The effector can be expressed only when both Cas9 protein and sgRNA are presented. (b)～(e) are schematic representations of the synthetic circuits (SC-1, SC-2, SC-3 and SC-4). The output genes were hRluc, hBAX, p21 and E-cadherin.

图3 与门微型基因线路的设计与构建［51］UPⅡ启动子驱动了Cas9 mRNA的转录，而TERT启动子被用来促进以LacI为目标的sgRNA的转录。输出的Renilla荧光素酶基因由LacI控制的CMV启动子调节。仅在UPⅡ启动子和TERT启动子都被激活时，荧光素酶基因才被表达。在迷你基因线路的设计中，UPⅡ和hTERT启动子被各自的转录因子结合元件所取代，c-Myc和Get1仅在膀胱癌细胞中同时有较高的表达水平。sgRNA1和sgRNA2初始表达后，进一步结合自身转录起始位点上游，通过正反馈机制扩增c-Myc和Get1的转录信号，分别扩增其下游基因的转录，此外，LacI基因被sgRNA2敲除，荧光素酶报告基因被转录激活。而在正常膀胱上皮细胞中，荧光素酶不能有效转录，并被背景水平表达的微量LacI进一步沉默

Fig. 3 Design and construction of the AND gate minigene circuits[51]The UPⅡ promoter drove the transcription of Cas9 mRNA, while the hTERT promoter was used to promote the transcription of sgRNA targeting LacI. The output Renilla luciferase gene was regulated by a LacI-controlled CMV promoter. The luciferase was expressed only when both UPⅡ promoter and TERT promoter were both activated. In the design of the minigene circuit, the UPⅡ and hTERT promoters were replaced by their respective transcription factor binding elements. Both c-Myc and Get1, only in bladder cancer cells, had a relative high expression level at the same time. After initial expression of sgRNA1 and sgRNA2, they could further bind upstream of their own transcription initiation sites and then amplify the transcription signals of c-Myc and Get1 through the positive feedback mechanism to amplify their corresponding downstream genes transcription, respectively. Furthermore, the LacI gene was knocked out by sgRNA2, and luciferase reporter gene was activated by transcription. In normal bladder epithelial cells, luciferase could not be effectively transcribed and was further silenced by trace amounts of LacI expressed at the background level

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CRISPR-Cas9系统在肿瘤生物学中的应用及前景

Application and prospect of CRISPR-Cas9 system in tumor biology

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