哺乳动物合成基因组学研究进展

doi:10.12211/2096-8280.2021-006

摘要/Abstract

摘要：

合成基因组学通过设计与合成大片段DNA序列，开展基因组尺度的工程化改造或从头合成，从而揭示基因型和表型的关联，并构建预定功能的生物。正如在大肠杆菌、酿酒酵母等低等模式生物中合成基因组学的实践，对哺乳动物基因组的大片段DNA设计再造也必然会加深对更为复杂的动物基因组的理解并加强对基因组的功能重塑。面对生命健康领域存在的重大挑战，设计合成哺乳动物大片段DNA为其提供了新的思路和解决方案，特别是在染色体疾病模型构建、人源化免疫系统等方面展示出独特的应用潜力。然而，目前大片段DNA在哺乳动物细胞中的设计和操纵仍是一个巨大的挑战，面临着高等哺乳动物基因组注释不完善、复杂序列组装困难、穿梭载体通用性差、大片段DNA转移低效等问题。本文围绕设计-组装-转移的技术路线，评述哺乳动物合成基因组学领域的重要研究进展，详细介绍重要的技术突破，并展望哺乳动物合成基因组学在医药健康领域的应用。

关键词: 合成基因组学, 基因组编写, 合成型哺乳动物染色体, 基因组工程, 哺乳动物细胞, 大片段DNA操纵技术

Abstract:

Synthetic genomics aims at genome-scale engineering or de novo synthesis through the design and chemical synthesis of large DNA sequences, which contributes to the revealing of connections between genotype and phenotype to construct organisms with expected functions. With the advances of synthetic genomics in lower model organisms, such as Escherichia coli and Saccharomyces cerevisiae, designing and rebuilding the large DNA fragments for mammalians could enhance the functional remodeling of their genomes. Designing higher mammalian genomes based on principles developed with the lower organism genome design can lead to understanding of more complex mammalian genomes, and in the meantime improve the design principles. In spite of multiple challenges, design and synthesis of mammalian large DNA fragments would provide promising methods and solutions. Overcoming the technical difficulties of manipulating large fragments of DNA in mammalian cells shows unique potentials in a variety of applications. For example, it can customize chromosomes for the construction of chromosomal disease models, build more complete humanized immune systems, etc. However, the current design and manipulation of large DNA fragments in mammalian cells are faced with many unsolved bottlenecks. Although the whole genome sequencing of several higher mammals has been completed, the annotation for the genomes of higher mammals is still far from complete. The existing assembly technology is difficult to accurately assemble complex repetitive sequences, and the vector presents poor versatility for shuttling between different cells. Moreover, the lower efficiency in transferring large fragments of DNA is a major bottleneck hindering the manipulation of large fragments in mammalian cells. This article systematically reviews recent progress in synthetic mammalian genomics by focusing on the breakthroughs in design-assembly-transfer technical route, and highlights further applications in human medicines and healthcare field.

Key words: synthetic genomics, genome writing, synthetic mammalian chromosome, genome engineering, mammalian cells, manipulation of large DNA

中图分类号:

何博, 付宗恒, 吴毅, 赵广荣. 哺乳动物合成基因组学研究进展[J]. 合成生物学, 2022, 3(1): 78-97.

Bo HE, Zongheng FU, Yi WU, Guangrong ZHAO. Research progress of synthetic mammalian genomics[J]. Synthetic Biology Journal, 2022, 3(1): 78-97.

图/表 4

图1 基因组的设计原则

Fig. 1 Design principles for genomes

图2 合成型哺乳动物染色体着丝粒区域（a）基于卫星（satellite）重复序列自下而上构建的着丝粒区域；（b）基于非天然重复序列自下而上构建的着丝粒区域；（c）自上而下截短的人工染色体

Fig. 2 Centromeric regions of synthetic mammalian chromosomes(a) Bottom-up centromere region based on satellite repeat sequence; (b) Bottom-up centromere region based on non-natural repetitive sequences; (c) Top-down artificial chromosomes from truncated natural chromosomes

表1 哺乳动物细胞大片段DNA的转移方法

Tab. 1 Method for delivering large fragments of DNA to mammalian cells

方法	转移介质	最大承载量	目的细胞	存在缺点	文献
脂质体转染	FuGENE 6	200 kb	HT1080	脂质材料对细胞有毒性	[46]
脂质体转染	Lipofection	400 kb	HT1080	脂质材料对细胞有毒性	[86]
电转	缓冲溶液	200 kb	mESc	需要昂贵细胞电转仪	[63, 87]
显微注射	缓冲溶液	590 kb	CHO	显微操作通量低	[88-89]
病毒载体	HSV-1	152 kb	hESc	操作步骤烦琐、存在生物安全问题	[90-91]
病毒载体	Epstein-barr virus	330 kb	Raji	操作步骤烦琐、存在生物安全问题	[92]
MMCT	微细胞	Mb级	HT1080	操作步骤烦琐	[93]
MMCT	微细胞	Mb级	Hela
Eco-MMCT	微细胞	Mb级	mESc
酵母原生质体-细胞融合	酵母原生质体	1.1 Mb	HEK293	操作步骤烦琐	[94]
		100 kb	HeLa		[95]
		1.4 Mb	mES

图3 哺乳动物细胞大片段DNA的转移方法（a）由酿酒酵母组装的DNA分子在大肠杆菌中富集后提取纯化到体外并通过显微注射、脂质体转染、电穿孔转移或病毒介导的转染等方法转移到宿主细胞中；（b）由酿酒酵母组装的DNA分子通过PEG介导的酵母原生质体与哺乳动物细胞融合转移到宿主细胞中；（c）在供体细胞中通过自上而下构建的人工染色体以微细胞介导的染色体转移方式转移到宿主细胞

Fig. 3 Method for delivering large fragments of DNA to mammalian cells(a) DNA molecules assembled by S.cerevisiae are enriched in E. coli, extracted and purified in vitro and transferred to host cells by microinjection, lipofection, electroporation, or virus-mediated transduction; (b) DNA molecules assembled by S.cerevisiae are transferred to host cells through the PEG-mediated fusion of yeast protoplasts and mammalian cells; (c) Top-down artificial chromosomes constructed in donor cell are transferred to host cell through the microcell-mediated chromosome transfer

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