Synthetic biology for next-generation genetic diagnostics

doi:10.12211/2096-8280.2022-061

Synthetic Biology Journal ›› 2023, Vol. 4 ›› Issue (2): 318-332.DOI: 10.12211/2096-8280.2022-061

• Invited Review • Previous Articles Next Articles

Synthetic biology for next-generation genetic diagnostics

Hailong LV¹^,²^,³, Jian WANG², Hao LV⁴, Jin WANG²^,⁵, Yong XU³, Dayong GU³

^1.Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging. School of Biomedical Engineering，School of Medicine，Shenzhen University，Shenzhen 518060，Guangdong，China
^2.Shenzhen Institute of Translational Medicine，Health Science Center，The First Affiliated Hospital of Shenzhen University，Shenzhen Second People's Hospital，Shenzhen 518035，Guangdong，China
^3.Department of Laboratory Medicine，The First Affiliated Hospital of Shenzhen University，Shenzhen Second People's Hospital，Shenzhen 518035，Guangdong，China
^4.Department of Neurosurgery，Shenzhen Key Laboratory of Neurosurgery，the Shenzhen Second People’s Hospital，First Affiliated Hospital of Shenzhen University，Shenzhen 518037，Guangdong，China
^5.Shanghai Tolo Biotechnology Co. ，Ltd. ，Shanghai 200233，China

Received:2022-11-02 Revised:2022-12-17 Online:2023-04-27 Published:2023-04-30
Contact: Dayong GU

合成生物学在下一代基因诊断技术中的应用进展

吕海龙¹^,²^,³, 王建², 吕浩⁴, 王金²^,⁵, 徐勇³, 顾大勇³

^1.深圳大学医学部生物医学工程学院，广东省生物医学信息检测与超声成像重点实验室，广东深圳 518060
^2.深圳市第二人民医院，深圳市转化医学研究院，广东深圳 518035
^3.深圳市第二人民医院检验科，深圳市医学检验分子诊断重点实验室，广东深圳 518035
^4.深圳市第二人民医院神经外科，深圳市神经外科重点实验室，广东深圳 518037
^5.上海吐露港生物科技有限公司，上海 200233

通讯作者: 顾大勇
作者简介:吕海龙（1988—），男，博士，博士后。研究方向为合成生物学及CRISPR分子诊断技术开发。E-mail： lvhailong8828@163.com
顾大勇（1972—），男，博士，主任技师，博士生导师、博士后合作导师。研究方向为生物芯片、生物传感技术。E-mail：wanhood@163.com
基金资助:
国家重点研发计划(2018YFA0903700);深圳市医疗卫生三名工程(SZSM202011017)

Abstract

Abstract:

In recent years, rapid advances have been made for synthetic biology technologies, which contribute greatly to technological innovations and applications in genetic diagnostics. Diagnostic methods for accurate detection of diseases and monitoring treatment responses are essential for effective clinical managements. Synthetic biology seeks to redesign biological systems to perform new functions in a predictable manner. A review on recent advances in synthetic biology as a diagnostic method for accurate detection of diseases and monitoring therapeutic responses is essential for effective clinical management, and also important for prevention, prediction and prognosis of diseases. In this article, we first describes the categories of synthetic biology applications in genetic diagnostics, including different biosensors that have been developed for both in vitro and in vivo monitoring. Subsequently, perspectives of the next generation of genetic diagnostic technologies and progress of synthetic biology devices and technologies in genetic diagnostics are highlighted. In addition, we further address the current technical challenges in genetic diagnostics and clinical applications. Finally, we draw attention to the latest innovations in synthetic biology that may have a significant impact on the future applications of genetic diagnostics.

Key words: synthetic biology, genetic diagnostics, CRISPR, translational medicine, biotechnology

摘要：

近年来，合成生物学技术取得了飞速的发展，与此同时也极大地推动了基因诊断领域的技术革新和应用拓展。准确诊断疾病和监测治疗后反应的检测方法对有效的临床管理至关重要，合成生物学作为一门以设计为导向的学科，试图重新设计生物系统，以便以可预测的方式执行新的功能，对这一领域的最新进展进行综述对疾病的预防、预测、诊断、治疗和预后具有重要意义。本文以该主题作为切入点，首先介绍了目前合成生物学在基因诊断中的应用类别，包括已经被开发出来的用于体外和体内的不同生物传感器；随后介绍了下一代基因诊断技术的发展方向以及基因诊断领域的合成生物学装置和技术进展；此外，本文讨论了目前基因诊断领域中存在的技术问题及其在临床应用中面临的挑战。最后，我们提请注意合成生物学中的最新创新，这些创新可能会对基因诊断的未来应用产生重大影响。

关键词: 合成生物学, 基因诊断, 成簇的规律间隔的短回文重复序列, 转化医学, 生物技术

CLC Number:

Q819

Hailong LV, Jian WANG, Hao LV, Jin WANG, Yong XU, Dayong GU. Synthetic biology for next-generation genetic diagnostics[J]. Synthetic Biology Journal, 2023, 4(2): 318-332.

吕海龙, 王建, 吕浩, 王金, 徐勇, 顾大勇. 合成生物学在下一代基因诊断技术中的应用进展[J]. 合成生物学, 2023, 4(2): 318-332.

Figures/Tables 6

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技术类型	方法	原理	应用对象	效果	优点	参考文献
环境污染领域的检测分析	基因修饰	大肠杆菌基因修饰合成可感知Cu²⁺并产生核黄素的孔蛋白，利用核黄素产量计算MFC生物传感器的最大电压，进而实现水中Cu²⁺的原位检测	水中Cu²⁺的原位检测	结果与现有的比色法、FAAS、ICP-OES等分析方法相一致	一种快速、经济的分析替代方案	[2]
环境污染领域的检测分析	双色荧光报告基因	在一个基因结构中使用双色荧光报告基因来检测生物可利用的镉，双色荧光的产生与镉的暴露浓度成正比	环境水样中的Cd（Ⅱ）	一种新型双传感、全细胞生物传感器的首次开发报告	可同时检测镉及其毒性效应	[3]
环境污染领域的检测分析	PCR，IFAT	PCR检测贾第虫、弓形虫和隐孢子虫属的DNA，免疫荧光抗体试验（IFAT）检测贾第虫和贾第虫属和隐孢子虫属的DNA的存在	饮用水中的原生生物	分析得出哥伦比亚的饮用水中致病性原生生物样本的流行率很高	两种检测方法相互验证	[4-5]
环境污染领域的检测分析	逆转录巢式PCR	开发RT-PCR引物集，优化甲型和乙型肝炎病毒（HAV和HEV）检测的标准模板	地下水中HAV和HEV	开发的巢式RT-PCR有望通过监测HAV和HEV来评估水的安全性	可区分假阳性，提高了检测效率	[6]
环境污染领域的检测分析	qPCR	采用HPLC-MS/MS测定抗生素，采用实时定量PCR定量，调查抗生素及其相应的ARGs的发生和分布	抗生素耐药性基因（ARGs）	人为活动及暴雨天气对抗生素污染和ARGs水平均有显著影响	增加对污水污染与ARG之间联系的了解	[7-8]
肠道微生物的检测分析	NO感应开关	设计NO响应生物传感器,在大肠杆菌Nisle 1917和迷你Sim Cells中对电路进行了表征和优化	NO	优化的基因电路NO检测限有所提高，基于Sim Cells的NO生物传感器可以用作合成生物学的安全传感器底盘	特征良好的硝酸传感系统有助于细菌治疗和合成生物学的快速发展	[9]
肠道微生物的检测分析	无细胞开关传感器，RT-qPCR	在基于纸张的无细胞反应中使用RNA开关传感器进行按需和简单的微生物组样本分析	肠道微生物群和宿主生物标志物	可以用于快速、廉价地检测毒素mRNA来诊断艰难梭菌感染	快速、廉价，比标准的基于DNA的qPCR诊断更灵敏	[10-11]
传染病诊断	酶报告基因	设计一种专门检测肠道中霍乱弧菌的群体感应信号乳杆菌菌株，并触发一种容易在粪便样本中检测到的酶报告基因的表达	霍乱弧菌	使用含有天然和工程乳杆菌菌株的发酵食品进行预防性饮食干预，可能会阻碍霍乱的进展	可提供快速保护，以抵御霍乱等快速进展的感染	[12]
传染病诊断	新型合成纳米体	通过筛选酵母表面显示的合成纳米体序列库，开发可破坏Spike和ACE2之间相互作用的纳米体	SARS-CoV-2病毒	纳米体mNb6-tri对Spike和SARS-CoV-2的感染具有中和作用	气溶胶介导的纳米体中和剂可以直接传递到气道上皮细胞	[13]
器官移植后检测	mRNA 疫苗	设计一种编码ZIKV结构基因的mRNA疫苗，编码prM-E基因产生病毒样颗粒，导致中和抗体滴度高，保护ZIKV感染并赋予杀菌免疫	ZIKV热病毒	改良的mRNA疫苗可以预防ZIKV疾病	可以降低个体对随后暴露于DENV的敏感性风险	[14]
器官移植后检测	酶抑制剂	抑制CMV DNA末端酶复合物的活性，阻止病毒DNA的加工和包装，从而发挥抗病毒作用	巨细胞病毒（CMV）	用于人类巨细胞病毒感染的治疗和预防	与单独使用免疫抑制剂相比有更好的耐受性	[15]
遗传病诊断	基因治疗	以基因矫正的热休克蛋白及其后代作为细胞载体，将分子传递到循环和组织中	自体造血干/祖细胞（HSPCs）	有可能治愈由血液系统发育功能改变引起的单基因遗传性疾病	具有更安全的整合谱	[16]
遗传病诊断	下一代基因测序（NGS）	通过针对所有鱼鳞病相关基因的多基因面板测试，使用NGS进行分子分析	中国人遗传学鱼鳞病	证明遗传性鱼鳞病是一组不同的角化症，扩大了中国人遗传性鱼鳞病的突变谱和临床表型	NGS技术极大提高了遗传性鱼鳞病的诊断效率	[17]
肿瘤基因诊断	工程免疫细胞	利用免疫细胞、核酸和细菌作为底盘的癌症基因电路疗法	肿瘤及癌症	合成生物学可创造出更有效的适应性疗法，使其能够特异性靶向癌细胞，同时保留健康细胞	可按需进行肿瘤及癌症的免疫治疗	[18-20]
基因突变的检测	原核生物核糖核酸调节剂	从头设计一种在体内和无细胞转录-翻译反应中提供超特异性RNA检测能力的原核核糖体调节因子	核糖体调节因子	开发用于与癌症、耐药性和遗传疾病相关的一系列突变的核糖体调节因子	实用，强大的分子探针	[21]

技术类型	方法	原理	应用对象	效果	优点	参考文献
环境污染领域的检测分析	基因修饰	大肠杆菌基因修饰合成可感知Cu²⁺并产生核黄素的孔蛋白，利用核黄素产量计算MFC生物传感器的最大电压，进而实现水中Cu²⁺的原位检测	水中Cu²⁺的原位检测	结果与现有的比色法、FAAS、ICP-OES等分析方法相一致	一种快速、经济的分析替代方案	[2]
环境污染领域的检测分析	双色荧光报告基因	在一个基因结构中使用双色荧光报告基因来检测生物可利用的镉，双色荧光的产生与镉的暴露浓度成正比	环境水样中的Cd（Ⅱ）	一种新型双传感、全细胞生物传感器的首次开发报告	可同时检测镉及其毒性效应	[3]
环境污染领域的检测分析	PCR，IFAT	PCR检测贾第虫、弓形虫和隐孢子虫属的DNA，免疫荧光抗体试验（IFAT）检测贾第虫和贾第虫属和隐孢子虫属的DNA的存在	饮用水中的原生生物	分析得出哥伦比亚的饮用水中致病性原生生物样本的流行率很高	两种检测方法相互验证	[4-5]
环境污染领域的检测分析	逆转录巢式PCR	开发RT-PCR引物集，优化甲型和乙型肝炎病毒（HAV和HEV）检测的标准模板	地下水中HAV和HEV	开发的巢式RT-PCR有望通过监测HAV和HEV来评估水的安全性	可区分假阳性，提高了检测效率	[6]
环境污染领域的检测分析	qPCR	采用HPLC-MS/MS测定抗生素，采用实时定量PCR定量，调查抗生素及其相应的ARGs的发生和分布	抗生素耐药性基因（ARGs）	人为活动及暴雨天气对抗生素污染和ARGs水平均有显著影响	增加对污水污染与ARG之间联系的了解	[7-8]
肠道微生物的检测分析	NO感应开关	设计NO响应生物传感器,在大肠杆菌Nisle 1917和迷你Sim Cells中对电路进行了表征和优化	NO	优化的基因电路NO检测限有所提高，基于Sim Cells的NO生物传感器可以用作合成生物学的安全传感器底盘	特征良好的硝酸传感系统有助于细菌治疗和合成生物学的快速发展	[9]
肠道微生物的检测分析	无细胞开关传感器，RT-qPCR	在基于纸张的无细胞反应中使用RNA开关传感器进行按需和简单的微生物组样本分析	肠道微生物群和宿主生物标志物	可以用于快速、廉价地检测毒素mRNA来诊断艰难梭菌感染	快速、廉价，比标准的基于DNA的qPCR诊断更灵敏	[10-11]
传染病诊断	酶报告基因	设计一种专门检测肠道中霍乱弧菌的群体感应信号乳杆菌菌株，并触发一种容易在粪便样本中检测到的酶报告基因的表达	霍乱弧菌	使用含有天然和工程乳杆菌菌株的发酵食品进行预防性饮食干预，可能会阻碍霍乱的进展	可提供快速保护，以抵御霍乱等快速进展的感染	[12]
传染病诊断	新型合成纳米体	通过筛选酵母表面显示的合成纳米体序列库，开发可破坏Spike和ACE2之间相互作用的纳米体	SARS-CoV-2病毒	纳米体mNb6-tri对Spike和SARS-CoV-2的感染具有中和作用	气溶胶介导的纳米体中和剂可以直接传递到气道上皮细胞	[13]
器官移植后检测	mRNA 疫苗	设计一种编码ZIKV结构基因的mRNA疫苗，编码prM-E基因产生病毒样颗粒，导致中和抗体滴度高，保护ZIKV感染并赋予杀菌免疫	ZIKV热病毒	改良的mRNA疫苗可以预防ZIKV疾病	可以降低个体对随后暴露于DENV的敏感性风险	[14]
器官移植后检测	酶抑制剂	抑制CMV DNA末端酶复合物的活性，阻止病毒DNA的加工和包装，从而发挥抗病毒作用	巨细胞病毒（CMV）	用于人类巨细胞病毒感染的治疗和预防	与单独使用免疫抑制剂相比有更好的耐受性	[15]
遗传病诊断	基因治疗	以基因矫正的热休克蛋白及其后代作为细胞载体，将分子传递到循环和组织中	自体造血干/祖细胞（HSPCs）	有可能治愈由血液系统发育功能改变引起的单基因遗传性疾病	具有更安全的整合谱	[16]
遗传病诊断	下一代基因测序（NGS）	通过针对所有鱼鳞病相关基因的多基因面板测试，使用NGS进行分子分析	中国人遗传学鱼鳞病	证明遗传性鱼鳞病是一组不同的角化症，扩大了中国人遗传性鱼鳞病的突变谱和临床表型	NGS技术极大提高了遗传性鱼鳞病的诊断效率	[17]
肿瘤基因诊断	工程免疫细胞	利用免疫细胞、核酸和细菌作为底盘的癌症基因电路疗法	肿瘤及癌症	合成生物学可创造出更有效的适应性疗法，使其能够特异性靶向癌细胞，同时保留健康细胞	可按需进行肿瘤及癌症的免疫治疗	[18-20]
基因突变的检测	原核生物核糖核酸调节剂	从头设计一种在体内和无细胞转录-翻译反应中提供超特异性RNA检测能力的原核核糖体调节因子	核糖体调节因子	开发用于与癌症、耐药性和遗传疾病相关的一系列突变的核糖体调节因子	实用，强大的分子探针	[21]

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Synthetic biology for next-generation genetic diagnostics

合成生物学在下一代基因诊断技术中的应用进展

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