Base editing technology and its application in microbial synthetic biology

doi:10.12211/2096-8280.2022-053

Abstract

Abstract:

The discovery and development of the CRISPR/Cas system have a revolutionary influence on life sciences. A series of tools derived from the CRISPR/Cas system have brought great convenience to research in the field of life sciences. The base editors developed based on the CRISPR/Cas system are gene editing tools that can achieve base conversions and transversion on target. The base editors are constructed by fusing cytosine or adenosine deaminase, and other functional elements to Cas proteins with abolished double strand DNA cleavage activity to convert cytidine or adenine into other bases at genome on-target sites guided by sgRNAs. Base editors have shown great potential in biology, medicine and related fields. Although they have already been continuously optimized, there are still problems affecting further application of base editors. In this review, we briefly describe the development of DNA base editors. Furthermore, we introduce in detail the problems of the limited editing range of base editors as well as the corresponding optimization strategies by increasing the target sites recognized by the locator moiety and expanding or narrowing the editing window of the effector moiety. At the same time, we introduce several off-target editing detection methods specially developed for base editing. Based on usual and developed detection methods, multiple and frequent off-target editing caused by base editors were found at both DNA and RNA levels. We also introduced various effective optimization strategies to improve the editing specificity of the base editors in every respect. Most of these strategies are based on protein modification, but also on optimization of sgRNA and spatio-temporal regulation of base editing systems. These measures greatly enrich the application scenarios of the base editors. Then, we discuss the progress on applying base editors to the field of microbial synthetic biology, including revealing the metabolic pathway and synthesis mechanism of natural products as well as improving the production of target compounds in multiple species. Finally, we envisage the promising development of base editing in synthetic biology in the future.

Key words: base editors, improvement of specificity, expansion of target range, microorganism, synthetic biology

摘要：

CRISPR/Cas系统的发现与发展对生命科学领域产生了革命性的影响，借助CRISPR/Cas系统研发出的一系列工具为相关领域的研究带来了极大的便利。碱基编辑器便是其中一类基于CRISPR/Cas系统开发的可实现碱基转换和颠换的基因编辑工具，其通过将胞苷或腺苷脱氨酶以及其他功能元件与失去双链切割活性的Cas蛋白相融合，由sgRNA引导，实现对基因组上目标位置胞嘧啶或腺嘌呤的碱基转换。碱基编辑器一经开发便在生物学、医学等领域展现出巨大的应用潜力，虽然经过不断优化，但目前在使用时仍然存在着许多制约因素。本文简述了几种主要碱基编辑器的发展，并介绍了碱基编辑器存在的靶向范围受限和脱靶编辑的问题以及现有的优化措施。同时列举了我国部分科研工作者将碱基编辑技术运用于微生物合成生物学领域的进展，并展望了碱基编辑技术的发展及其在微生物合成生物学领域的应用前景。

关键词: 碱基编辑器, 特异性优化, 靶向范围拓展, 微生物, 合成生物学

CLC Number:

Q812

WANG Yannan, SUN Yuhui. Base editing technology and its application in microbial synthetic biology[J]. Synthetic Biology Journal, 2023, 4(4): 720-737.

王雁南, 孙宇辉. 碱基编辑技术及其在微生物合成生物学中的应用[J]. 合成生物学, 2023, 4(4): 720-737.

Figures/Tables 1

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碱基编辑器	版本	组成	PAM (5′-3′)	特点	参考文献
CBE	BE3	rAPOBEC1-SpnCas9-UGI	NGG	初代碱基编辑器，TC基序偏好性	[7]
	Target-AID	SpnCas9-CDA1-UGI	NGG	初代碱基编辑器	[8]
	hA3A-BE3	hAPOBEC3A-SpnCas9-UGI	NGG	可编辑甲基化的C及GC基序的C	[9]
	BE4-Max	rAPOBEC1-SpnCas9-UGI-UGI	NGG	密码子优化，高活性	[10]
	dCpf1-BE	rAPOBEC1-dLbCpf1-UGI	TTTV	识别富含T的PAM	[11]
	Target-AID-NG	SpnCas9 (NG)-CDA1-UGI	NG	识别NG PAM	[12]
	A3A-PBE	hAPOBEC3A-SpnCas9-UGI	NGG	1～17号位编辑窗口，几乎无基序偏好性	[13]
	BE-PLUS	SunTag-SpnCas9-scFv-rAPOBEC1-UGI	NGG	4～16号位编辑窗口，高保真	[14]
	A3G-BEs	hAPOBEC3G-SpnCas9-UGI-UGI	NGG	编辑CC基序中的第二个C	[15]
	BE-PAPAPAP	rAPOBEC1-SpnCas9-UGI	NGG	5～6号位编辑窗口，PAPAPAP替换XTEN linker	[16]
	HF-BE3	rAPOBEC1-HFnCas9-UGI	NGG	减少sgRNA依赖的脱靶编辑	[17]
	YE1-BE4	rAPOBEC1 (W90Y, R126E)-SpnCas9-UGI-UGI	NGG	缩小编辑窗口，减少非sgRNA依赖的脱靶编辑	[18]
	SECURE BE3	rAPOBEC1 (R33A, K34A)-SpnCas9-UGI	NGG	减少RNA层面的脱靶编辑，缩小编辑窗口	[19]
	DdCBEs	TALE-split DddAtox-UGI	None	同时编辑DNA双链，可编辑线粒体、叶绿体基因组	[20]
	ZFDs	ZFP-split DddAtox-UGI	None	同时编辑DNA双链，可编辑线粒体、叶绿体基因组	[21]
ABE	ABE7.10	ecTadA-ecTadA*-SpnCas9	NGG	初代碱基编辑器	[22]
	ABEmax	ecTadA-ecTadA*-SpnCas9	NGG	密码子优化，高活性	[10]
	ABE8e	ecTadA-ecTadA*-SpnCas9	NGG	高活性，TadA-8e	[23]
	TaC9-ABE	ecTadA-ecTadA*-TALE,SpnCas9	NGG	消除sgRNA依赖的脱靶编辑	[24]
	ABE-nSpCas9-DS	ecTadA-ecTadA-SpnCas9 (DS)	NGG	拓宽编辑窗口，减少RNA层面的脱靶编辑	[25]
ACBE	A&C-BEmax	rAPOBEC1/hAID-ecTadA-ecTadA*-SpnCas9-UGI-UGI	NGG	可同时编辑A或C	[26]
	sgBE	SpnCas9, MCP-cytosine/adenosine deaminase	NGG	可同时编辑A或C	[27]
GBE	CGBE1	eUNG-rAPOBEC1 (R33A)-SpnCas9-UGI-UGI	NGG	可实现C变G的碱基颠换	[28]
	CGBE	rAPOBEC1-SpnCas9-rXRCC1	NGG	利用碱基切除修复通路实现C变G的碱基颠换	[29]

碱基编辑器	版本	组成	PAM (5′-3′)	特点	参考文献
CBE	BE3	rAPOBEC1-SpnCas9-UGI	NGG	初代碱基编辑器，TC基序偏好性	[7]
	Target-AID	SpnCas9-CDA1-UGI	NGG	初代碱基编辑器	[8]
	hA3A-BE3	hAPOBEC3A-SpnCas9-UGI	NGG	可编辑甲基化的C及GC基序的C	[9]
	BE4-Max	rAPOBEC1-SpnCas9-UGI-UGI	NGG	密码子优化，高活性	[10]
	dCpf1-BE	rAPOBEC1-dLbCpf1-UGI	TTTV	识别富含T的PAM	[11]
	Target-AID-NG	SpnCas9 (NG)-CDA1-UGI	NG	识别NG PAM	[12]
	A3A-PBE	hAPOBEC3A-SpnCas9-UGI	NGG	1～17号位编辑窗口，几乎无基序偏好性	[13]
	BE-PLUS	SunTag-SpnCas9-scFv-rAPOBEC1-UGI	NGG	4～16号位编辑窗口，高保真	[14]
	A3G-BEs	hAPOBEC3G-SpnCas9-UGI-UGI	NGG	编辑CC基序中的第二个C	[15]
	BE-PAPAPAP	rAPOBEC1-SpnCas9-UGI	NGG	5～6号位编辑窗口，PAPAPAP替换XTEN linker	[16]
	HF-BE3	rAPOBEC1-HFnCas9-UGI	NGG	减少sgRNA依赖的脱靶编辑	[17]
	YE1-BE4	rAPOBEC1 (W90Y, R126E)-SpnCas9-UGI-UGI	NGG	缩小编辑窗口，减少非sgRNA依赖的脱靶编辑	[18]
	SECURE BE3	rAPOBEC1 (R33A, K34A)-SpnCas9-UGI	NGG	减少RNA层面的脱靶编辑，缩小编辑窗口	[19]
	DdCBEs	TALE-split DddAtox-UGI	None	同时编辑DNA双链，可编辑线粒体、叶绿体基因组	[20]
	ZFDs	ZFP-split DddAtox-UGI	None	同时编辑DNA双链，可编辑线粒体、叶绿体基因组	[21]
ABE	ABE7.10	ecTadA-ecTadA*-SpnCas9	NGG	初代碱基编辑器	[22]
	ABEmax	ecTadA-ecTadA*-SpnCas9	NGG	密码子优化，高活性	[10]
	ABE8e	ecTadA-ecTadA*-SpnCas9	NGG	高活性，TadA-8e	[23]
	TaC9-ABE	ecTadA-ecTadA*-TALE,SpnCas9	NGG	消除sgRNA依赖的脱靶编辑	[24]
	ABE-nSpCas9-DS	ecTadA-ecTadA-SpnCas9 (DS)	NGG	拓宽编辑窗口，减少RNA层面的脱靶编辑	[25]
ACBE	A&C-BEmax	rAPOBEC1/hAID-ecTadA-ecTadA*-SpnCas9-UGI-UGI	NGG	可同时编辑A或C	[26]
	sgBE	SpnCas9, MCP-cytosine/adenosine deaminase	NGG	可同时编辑A或C	[27]
GBE	CGBE1	eUNG-rAPOBEC1 (R33A)-SpnCas9-UGI-UGI	NGG	可实现C变G的碱基颠换	[28]
	CGBE	rAPOBEC1-SpnCas9-rXRCC1	NGG	利用碱基切除修复通路实现C变G的碱基颠换	[29]

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