基因编辑技术的研究进展

doi:10.11843/j.issn.0366-6964.2025.01.001

摘要/Abstract

摘要：

基因编辑技术是修饰特定基因的新型分子工具，可在基因序列中有效引入新的突变，为分析基因功能和与表型相关的DNA序列提供了有效的手段。自发现以来，基因编辑技术已经成功应用在各种动物、植物及其他生物中，为基因治疗及精准作物育种等领域提供了重要技术支撑。本文旨在回顾近年来基因编辑工具的发展，阐述了经典编辑工具、碱基编辑工具和其他新编辑工具的进展，以期为相关领域科研人员进一步了解、优化编辑系统提供参考。

关键词: 基因编辑, ZFN编辑, TALENs编辑, CRISPR-Cas, 碱基编辑

Abstract:

Gene editing technology is a novel molecular tool for modifying specific genes, which can effectively introduce new mutations into gene sequences, providing effective means for analyzing gene function and the phenotype-related DNA sequences. Since its discovery, gene editing technology has been successfully applied in various animals, plants, and other organisms, providing important technical support for gene therapy and precision crop breeding. This article aims to review the development of gene editing tools in recent years, elucidate the progress of classic editing tools, base editing tools, and other new editing tools, in order to provide reference for researchers in related fields to further understand and optimize editing systems.

Key words: gene editing, ZFN editing, TALENs editing, CRISPR-Cas, base editing

中图分类号:

S813.1

包斌武, 邹惠影, 李俊良, 高晨, 高会江, 杜振伟, 张博玉, 李俊雅, 高雪. 基因编辑技术的研究进展[J]. 畜牧兽医学报, 2025, 56(1): 1-14.

BAO Binwu, ZOU Huiying, LI Junliang, GAO Chen, GAO Huijiang, DU Zhenwei, ZHANG Boyu, LI Junya, GAO Xue. Research Progress in Gene Editing Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 1-14.

图/表 6

图 1

表 1

CRISPR-Cas技术的发展与优化"

名称 Name	类型 Type	尺寸(AA) Size	特征 Feature	年份 Year	文献 Reference
SpCas9	Cas9	1 368	第一个在人类细胞中实现靶向诱变的Cas9同源物	2013	Cong等^[28]
SaCas9	Cas9	1 053	具有高效、特异性和耐受性良好	2015	Ran等^[31]
Cpf1	Cas12a	1 353	Cpf1只需要42 nt crRNA，切割导致原间隔区远端5′突出，提高基于NHEJ的基因插入的效率，一种单RNA引导的核酸酶，只需要crRNA，并包含单个RuvC结构域	2015	Zetsche等^[32]
xCas9	Cas9	1 368	扩大了PAM兼容性，DNA特异性比SpCas9高得多，脱靶活性低	2018	Hu等^[33]
Cas13a(C2c2)	Cas13	—	C2c2仅作为RNA引导的RNA靶向CRISPR效应子发挥作用	2016	Abudayyeh等^[34]
CjCas9	Cas9	984	CjCas9由984个氨基酸残基组成(比SpCas9或SaCas9小)	2017	Kim等^[35]
SpCas9-NG	Cas9	1 368	SpCas9-NG在NGG位点的裂解活性低于SpCas9, 使NG PAM识别成为可能	2018	Nishimasu等^[36]
LbCpf1 and FnCpf1	Cas12	1 300	由一个RuvC核酸内切酶结构域组成，在植物细胞中，FnCpf1编辑了一个TTV PAM位点，对Cpf1 TTTV PAM位点进行优化	2018	Zhong等^[37]
AaCas12b	Cas12	1 129	尺度小，基因组靶向范围广，不易脱靶，可以在体外4 ℃和100 ℃之间切割靶DNA	2018	Teng等^[38]
CasX(Cas12e)	Cas12	986	分子量小，编辑效率高，非特异性切割活性低	2019	Liu等^[39]
BhCas12b	Cas12	1 108	优化后的BhCas12 v4可以在各种基因组编辑环境中用作有效的可编程核酸酶	2019	Strecker等^[40]
SpG和SpRY	Cas9	140	SpRY核酸酶和碱基编辑器变体可以靶向几乎所有PAM，是目前最与PAM序列兼容的Cas9突变体	2020	Walton等^[41]
CasΦ(Cas12j)	Cas12	700~800	CasΦ体积小，但功能齐全，将多种功能组合成一个单一蛋白可实现更容易的载体介导递送，能更广泛的识别基因序列	2020	Pausch等^[42]
MAD7(ErCas12a)	Cas12	1 263	MAD7脱靶概率较低，具有不同的PAM识别位点，并且不需要反式激活CRISPR RNA(tracrRNA)	2021 2020 2020	Jarczynska等^[43] Liu等^[44] Price等^[45]
AtCas9	Cas9	—	突破PAM限制，实现近乎无PAM的切割	2022	Shi等^[46]
Casπ (Cas12l)	Cas12	850~867	通过识别CCN PAM来切割底物DNA，能在哺乳动物细胞中实现有效的基因编辑	2023	Sun等^[47]
Cas8-HNH和 Cas5-HNH	Cas8 Cas5	—	具有高度特异性，使用长达32个碱基对的gRNA，减少脱靶可能性	2023	Altae-tran等^[48]
AsCas12f1	Cas12	422	低毒性，高效率链霉菌基因编辑工具	2024	Hua等^[49]

表 1

表 2

碱基编辑系统的发展与优化"

类型 Type	名称 Name	特征 Feature	编辑效率 Editing efficiency	年份 Year	文献 Reference
BE编辑系统 BE editing system	BE1	由dCas9与APOBEC1的融合蛋白构成	表观编辑效率44%(体外)，0.8~7.7%(体内)	2016	Komor等^[50]
	BE2	将来源于噬菌体的抑制子UGI引入BE1	表观编辑效率20%(体内)	2016	Komor等^[50]
	BE3	将BE2中的dCas9替换为nCas9(D10A)	表观编辑效率20~30%(体外)，15~75%(体内)	2016	Komor等^[50]
	HF-BE3	将BE3与一种高保真SpCas9变体(HF-Cas9)结合	将BE3的脱靶水平降低了37倍	2017	Rees等^[64]
	BE4	优化nCas9与APOBEC1的Linker长度	表观编辑效率50%(体内)，效率比BE3提高1.5倍	2017	Komor等^[65]
	BE4max	BE4基础上优化核定位信号，密码子	效率比BE4提高3倍	2018	Koblan等^[53]
	ABE	成功实现了A-T到G-C的碱基更改	—	2017	Gaudelli等^[5]
	ABE7.10	野生型ecTadA单体与定向进化出的ecTadA* 单体融合形成单链异二聚体	编辑效率可达53%	2017	Gaudelli等^[5]
	ABEmax	通过对ABE7.10进行密码子优化以及增加NLS个数	编辑效率比ABE7.10提高~1.5至2倍	2018	Koblan等^[53]
	ABE8s	在BE7.10的TadA中引入额外的突变，构建了ABE8s	编辑效率比ABE7.10提高约4.2倍	2020	Gaudelli等^[54]
	ABE9	实现了高精度，低脱靶的碱基编辑	—	2023	Chen等^[66]
	hyABE	提高了靠近PAM区A-G的转换效率，扩大了编辑窗口	编辑效率43.0~94.6%	2023	Xue等^[67]
	GhABE8e	与ABE7.10相比，编辑效率更高，脱氨速度更快	平均编辑效率为60%~99.9%	2024	Wang等^[68]
	DAF-CBE和DAF-TBE	不依赖脱氨酶，扩展了碱基编辑器的转换类型，效率相似，但尺寸更小，脱靶效应更低	DAF-CBE平均编辑效率为20.7%； DAF-TBE平均编辑效率为22.5%	2024	Ye等^[69]
	GBE	在哺乳动物细胞中实现C-G的颠换，而在细菌中实现C-A的颠换	大肠杆菌中编辑效率为87.2%±6.9%；哺乳动物中编辑效率在5.3%~53.0%	2021	Zhao等^[70]
PE编辑系统 PE editing system	PE1	野生型的M-MLV逆转录酶与nCas9(H840A)的融合蛋白	对点突变的编辑效率最高可达到5.5%，小片段插入删除编辑效率在4%~17%之间	2019	Anzalone等^[6]
	PE2	对PE1的逆转录酶进行改造	点突变的编辑编辑效率相比PE1提高了1.6~5.1倍	2019	Anzalone等^[6]
	PE3	在pegRNA下游增加未编辑基因组DNA的识别位点	—	2019	Anzalone等^[6]
	PE4/PE5	PE2/PE3系统分别插入MMR抑制蛋白 PE4(PE2+MLH1dn)PE5(PE3+MLH1dn)	与PE2/PE3相比，将替换、小插入和小缺失引物编辑的效率分别平均提高7.7和2.0倍	2021	Chen等^[57]
	PEmax	通过改变RT密码子使用、SpCas9突变、NLS序列以及 nCas9和RT之间肽连接子的长度和组成来优化PE2蛋白	编辑效率大于30%	2021	Chen等^[57]
	PE6a-g	产生了比最先进编辑PEmax少516~810个碱基对的先导编辑	PE6变体的平均编辑效率比PEmax提高了1.4倍	2023	Doman等^[71]
	PE7	将La蛋白的N端结构域融合到现有PEmax C端(PEmax-C)	与PEmax相比，PE7编辑效率提高并对脱靶编辑影响最小	2024	Yan等^[58]
	CPE	优先识别富含T的基因组区域，可进行多基因的编辑，且脱靶效应极低	niCPE和sniCPE在人类细胞中的编辑频率分别为24.89%和40.75%	2024	Liang等^[59]
线粒体编辑系统 Mitochondrial editing system	DdCBE	成功实现了线粒体中C to T的碱基编辑	编辑效率15%~30%	2022	Mok等^[72]
	TALED	成功实现了线粒体中A to G的碱基编辑	—	2022	Cho等^[73]
	mitoBEs	不依赖于DddA，提升了线粒体碱基编辑的精准性	编辑效率为18%~36%	2023	Yi等^[63]
	CyDENT	细胞核、线粒体和叶绿体中均可实现高效胞嘧啶碱基编辑；不依赖CRISPR	14%的编辑效率和95%的链特异性	2023	Hu等^[74]

表 2

图 2

图 3

图 4

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