Advances in Large DNA Assembly and Transfer Based on <i>Saccharomyces cerevisiae</i>

doi:10.13523/j.cb.2203026

China Biotechnology

2022, Vol. 42

Issue (7): 101-112 DOI: 10.13523/j.cb.2203026

Advances in Large DNA Assembly and Transfer Based on Saccharomyces cerevisiae

Fang-fang TIAN^1,²,Bo HE^1,²,Yi WU^1,^2,^**(

)

1. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
2. Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin 300072, China

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Abstract

DNA assembly and transfer techniques are one of the core enabling technologies for synthetic biology. The increasing degree of complexity in the design and modification of living organisms has led to a growing demand for large DNA assembly and transfer methods. Nowadays,the assembly and transfer techniques of small DNA are well developed, while the manipulation of large DNA in vitro is complicated and inefficient due to DNA high molecular weight and ease to break. This review focuses on advances in large DNA assembly and transfer techniques in Saccharomyces cerevisiae and transfer technology. The methods of one-step assembly and iterative assembly in S. cerevisiae are introduced in detail. The transfer methods from the aspect of transfer in and out are highlighted, and researchers can better understand and choose these methods. In addition, the authors envisage that it is possible to make S. cerevisiae become a universal platform for assembly and transfer of large DNA, which enables large-scale genomic design and modification of more organisms.

Key words： Large DNA assembly Large DNA transfer Saccharomyces cerevisiae

Received: 11 March 2022 Published: 03 August 2022

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Corresponding Authors: Yi WU E-mail: yi.wu@tju.edu.cn

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Cite this article:

Fang-fang TIAN,Bo HE,Yi WU. Advances in Large DNA Assembly and Transfer Based on Saccharomyces cerevisiae. China Biotechnology, 2022, 42(7): 101-112.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2203026 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I7/101

Fig.1 Factors of import-assembly-export DNA fragments in S. cerevisiae

Fig.2 Schematic diagram of the CasHRA method(a)and schematic diagram of synX construction by SwAP-In(b)

分类		方法	原理	应用	组装大小	引文
酵母一次组装	组装成质粒形式	DNA组装器(DNA assembler,TAR)	多个待组装的DNA片段(两侧有同源序列)与载体在酿酒酵母体内直接利用同源重组形成环形质粒	多基因调控的生化途径组装,途径文库构建	约50 kb	[40][41-43][44-47]
		结合Golden Gate(VEGAS)	将启动子、基因、终止子在体外用Golden Gate组装成转录单元模块,再在酿酒酵母体内组装多模块	模块化组装多个转录单元,构建途径文库	约50 kb	[51]
		结合Gibson	将商业合成的短片段用多次Gibson组装至百kb,再通过酿酒酵母组装	完整基因组	约583 kb	[50]
		结合蓝白斑筛选(RADOM)	酿酒酵母组装后混合质粒通过大肠杆菌蓝白斑筛选减轻筛选工作	快速构建3~10 kb的DNA片段	3~10 kb	[52]
	靶向整合至基因组	内切核酸酶辅助(I-SceI-assisted integration)	用I-SceI在目标染色体基因座处引入双链断裂,从而促进组装构建体的整合	基因组上的多基因生化途径组装	约35 kb	[53]
		CRISPR/Cas9辅助(CasEMBLR/CrEdit/Di-CRISPR等)	CRISPR/Cas9系统在基因组多位点同时引入双链断裂,促进多位点同时组装	基因组上多位点无标记的多基因生化途径组装	-	[54] [57-59]
酵母迭代组装		内切核酸酶辅助(reiterative recombination)	用两种内切核酸酶HO和I-SceI,设计两种正交切割质粒,交替使用它们在基因组上引入双链断裂,逐步将DNA片段靶向整合至基因组	基因组上多基因途径文库的构建	-	[62]
		CRISPR/Cas9辅助(CasHRA)	通过原生质体融合将大质粒共同导入单个细胞,再利用CRISPR/Cas9系统释放出线性DNA进行同源重组组装	大肠杆菌基因组	约1 Mb	[60]
		逐步转换营养标签组装(SwAP-In)	在待组装DNA片段一侧设计不同的营养标签,每一次组装时都用新的营养标签替换先前的营养标签,通过直接筛选营养标签获得组装正确的菌株	合成型酵母染色体synII、synIII、synV、synVI、synX组装	约770 kb、约273 kb、约536 kb、约243 kb、约707 kb	[3-9]
		酵母交配、减数分裂重组介导的组装(MRA)	利用二倍体中减数分裂时期姐妹染色单体发生交叉互换的可能性进行组装	人源TCRαβ基因座、合成型染色体synXII	约1 Mb	[7, 61]

Table 1 DNA assembly method based on homologous recombination in S.cerevisiae

分类		方法	转移介质	转移大小	优点	缺点	引文
导入	化学方法	PEG-醋酸锂转化	PEG3350/LiOAc	约50 kb	可同时导入多片段,技术成熟	转移尺度小	[26]
		PEG-原生质体转化	PEG8000	约200 kb	可同时导入多片段,转移尺度较大	操作复杂,效率低	[66]
		阳离子聚合物包埋	阳离子聚合物	约15 kb	能使带负电荷的DNA分子聚集	通用性差,效率低	[67-68]
	物理方法	酵母原生质体电穿孔转化	山梨醇溶液	约200 kb	操作简单,转移尺度大	酵母致死率高	[69]
		完整酵母电穿孔转化	缓冲溶液	约15 kb	操作简单,不需要制备原生质体	酵母致死率高	[70]
		基因枪	—	约5 kb	直接导入至细胞核	需要昂贵的精密仪器,效率低	[71-73]
	生物方法	酵母-酵母原生质体融合	酵母原生质体	Mb级别	转移尺度大,不需要体外操作	供体酵母基因组可能干扰受体基因组,效率低	[60]
		酵母交配(mating)	—	Mb级别	转移尺度大,不需要体外操作	局限于酵母之间	[7, 61]
		细菌-酵母原生质体融合	酵母原生质体	Mb级别	转移尺度大,不需要体外操作	效率低	[74-75]
导出	从酵母中提取出后递送至其他宿主	电穿孔转移	常规方法提取,通过缓冲溶液电转	约200 kb	可用于其他方法难以转染的细胞系	需要昂贵的细胞电转仪,难操作	[85]
		脂质体转染	常规方法提取,通过脂质体转染	约300 kb	有商业化的试剂盒	脂质材料对细胞有毒性	[88-89]
		显微注射转移	琼脂糖包埋法提取纯化后注射	约600 kb	转移尺度大,直接转到细胞器(线粒体)或细胞核中	无法高通量操作,对细胞伤害大,需要昂贵的激光设备	[86-87]
		阳离子聚合物包埋转移	琼脂糖包埋法提取纯化后通过阳离子聚合物转移	Mb级别	可进行大尺度DNA转移,不需要高级设备	重复性差	[90]
	从酵母中直接递送至目的宿主	酵母交配(mating)	—	Mb级别	转移尺度大,不需要体外操作	局限于酵母之间	[94-95]
	从酵母中直接递送至目的宿主	酵母原生质体-哺乳动物细胞融合	酵母原生质体	Mb级别	转移尺度大,不需要体外操作	供体酵母基因组可能干扰受体基因组,效率低	[96-99]

Table 2 Large DNA transfer methods based on S.cerevisiae


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