基于CRISPR/Cas9的基因编辑技术研究进展及其在植物中的应用

doi:10.11983/CBB22020

植物学报 ›› 2022, Vol. 57 ›› Issue (4): 508-531.DOI: 10.11983/CBB22020

基于CRISPR/Cas9的基因编辑技术研究进展及其在植物中的应用

何晓玲, 刘鹏程, 马伯军, 陈析丰()

浙江师范大学化学与生命科学学院, 金华 321004

收稿日期:2022-01-21 修回日期:2022-04-24 出版日期:2022-07-01 发布日期:2022-07-14
通讯作者: 陈析丰
作者简介:第一联系人：^† 共同第一作者
基金资助:
国家自然科学基金(31871605);国家自然科学基金(32071987);国家自然科学基金(32101697);浙江省自然科学基金(LD19C130001);浙江省自然科学基金(Q22C133851)

Advance in Gene-editing Technology Based on CRISPR/Cas9 and Its Application in Plants

He Xiaoling, Liu Pengcheng, Ma Bojun, Chen Xifeng()

College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China

Received:2022-01-21 Revised:2022-04-24 Online:2022-07-01 Published:2022-07-14
Contact: Chen Xifeng
About author:* E-mail: xfchen@zjnu.cn
First author contact:^† These authors contributed equally to this paper

摘要/Abstract

摘要： CRISPR/Cas9技术是利用RNA靶向引导Cas9核酸酶对基因组中的目标基因进行编辑的生物技术。近年来, 该技术的多种新型基因编辑器更新迅猛, 编辑效果愈加精细和高效, 在作物定向分子设计育种中展现出巨大的应用前景。该文对CRISPR/Cas9及其相关编辑器的技术原理、编辑效果和应用情况进行综述, 并探讨了该技术在应用中面临的问题、应对措施和发展前景, 旨在为相关领域的科研工作者提供参考。

关键词: CRISPR/Cas9, 基因编辑, 技术进展, 在植物中应用

Abstract: CRISPR/Cas9 technology is a gene editing strategy using Cas9 nuclease guided by RNA to target an interest gene in genome. Recently, a large number of new-type gene editors based on the CRISPR/Cas9 have been updated rapidly and become more precisely and efficiently in gene editing, which has a great application prospect in crop molecular-designing breeding. This paper summarized the technical principles, editing effects and applications of the CRISPR/cas9 and its related gene editors, and also discussed the aspects of dilemmas, countermeasures and prospects, intending to provide reference for scientific researchers in the related fields.

Key words: CRISPR/Cas9, gene editing, technical progress, application in plants

何晓玲, 刘鹏程, 马伯军, 陈析丰. 基于CRISPR/Cas9的基因编辑技术研究进展及其在植物中的应用. 植物学报, 2022, 57(4): 508-531.

He Xiaoling, Liu Pengcheng, Ma Bojun, Chen Xifeng. Advance in Gene-editing Technology Based on CRISPR/Cas9 and Its Application in Plants. Chinese Bulletin of Botany, 2022, 57(4): 508-531.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22020

https://www.chinbullbotany.com/CN/Y2022/V57/I4/508

图/表 9

表1 各类碱基编辑器的特点

Table 1 Characteristics of all kinds of base editors

编辑技术	编辑器	脱氨酶/逆转录酶		Cas蛋白	PAM (5′-3′)		活性窗口	编辑类型		特点	参考文献
胞嘧啶碱基编辑(CBEs)	BE3	rAPOBEC1		nCas9^D10A	NGG		C₄-C₈	C:G>T:A		实现C>T替换, 无需DSB或提供模板	Komor et al., 2016
	nCas9-PBE	rAPOBEC1		nCas9^D10A	NGG		C₃-C₉			在植物中实现精准高效的C>T替换	Zong et al., 2017
	Target-AID	PmCDA1		nCas9^D10A	NGG		C₁-C₅			具有较好的GC碱基编辑能力	Nishida et al., 2016
	dCas9-AIDx	hAID		dCas9	NCN		C₄-C₉			具有较好的GC碱基编辑能力	Ma et al., 2016
	A3A^Y130F- CBE_V01 A3A^Y130F- CBE_V04	hAPOBEC3A^Y130F		nCas9^D10A	NGG		C₄-C₁₅			活性窗口拓宽至12 nt	Ren et al., 2021a
	eCDAL	LjCDA1L-4		nCas9^D10A	NGG		C₁-C₁₂			活性窗口拓宽至12 nt	Xu et al., 2021
	BE3-PAPAPAP	rAPOBEC1		nCas9^D10A	NGG		C₅-C₆			活性窗口缩小至1-2 nt	Tan et al., 2019
	PhieCBEs	evorAPOCBEC1 evoFERNY evoCDA1 hA3A		nCas9-NG eCas9n- NG	NG NGG		C_-1-C₁₅			编辑窗口广	Zeng et al., 2020
	BE3^R126E BE3^R132E YE1-BE3	rAPOBEC1^R126E rAPOBEC1^R132E rAPOBEC1^W90Y+R126E		nCas9^D10A	NGG		C₅-C₇			脱靶率显著降低	Zuo et al., 2020
腺嘌呤碱基编辑(ABEs)	ABE7.10	ecTadA:ecTadA^*		nCas9^D10A	NGG		A₄-A₇	A:T>G:C		实现A>G替换, 无需DSB或提供模板	Gaudelli et al., 2017
	PABE	ecTadA:ecTadA^*		nCas9^D10A	NGG		A₄-A₈			在植物中实现精准高效的A>G替换	Li et al., 2018b
	ABE-Ps	ecTadA:ecTadA^*		nCas9^D10A SaCas9	NGG NNNRRT		A₁-A₁₅			在植物中实现精准高效的A>G替换	Hua et al., 2018
	rBE14	ecTadA:ecTadA^*		nCas9^D10A	NGG		A₅-A₇			荧光检测编辑植株	Yan et al., 2018
	rABE8e	TadA8e^V106W		nCas9^D10A nCas9-NG	NG NGG		A₅-A₆			极高的靶点编辑效率和碱基纯合替换效率	Wei et al., 2021
	PhieABEs	TadA8e		nCas9-NG SpGn SpRYn	NG NGN NNN		A₁-A₁₄			编辑效率高, 几近无PAM靶向, 窗口广	Tan et al., 2022
	SpRY-ABE8e	ecTadA:ecTadA^*		nCas9^D10A	NNN		A₃-A₁₀			几近无PAM靶向	Ren et al., 2021b
	ABE7.10^F148A	ecTadA^F148A: ecTadA^*F148A		nCas9^D10A	NGG		A₅-A₆			活性窗口缩小至1-2 nt	Zhou et al., 2019
CG碱基编辑(GBEs)	GBE	AID rAPOBEC1		nCas9^D10A	NGG		C₃-C₇	C:G>A:T/G:C		实现嘧啶与嘌呤间的颠换	Zhao et al., 2021
CG碱基编辑(GBEs)	CGBE	rAPOBEC1		nCas9^D10A	NGG		C₅-C₆	C:G>A:T/G:C		实现嘧啶与嘌呤间的颠换	Chen et al., 2021
双碱基编辑(A&CBE)	A&C-BEmax	hAID ecTadA:ecTadA^*		nCas9^D10A	NGG		C₂-C₁₇ A₄-A₇	C:G>T:A A:T>G:C		实现A、C共编辑	Zhang et al., 2020
	Target-ACE	PmCDA1 ecTadA:ecTadA^*			NGG		C₁-C₁₀ A₄-A₈				Sakata et al., 2019
	SPACE	PmCDA1 ecTadA^*			NGG		C₂-C₇ A₄-A₇				Grünewald et al., 2020
	ACBE	PmCDA1 ecTadA:ecTadA^*			NGG		C₁-C₇ A₄-A₆				Xie et al., 2020
	STEME	hAPOBEC3A ecTadA:ecTadA^*			NG NGD		C₁-C₁₇ A₄-A₈			在植物中实现A、 C共编辑	Li et al., 2020a
	pDuBE1	TadA8e			NGG		C₁-C₁₀ A₂-A₉			在植物中实现A、 C共编辑	Xu et al., 2021
编辑技术	编辑器		脱氨酶/逆转录酶	Cas蛋白	PAM (5′-3′)	活性窗口			编辑类型	特点	参考文献
先导编辑(PEs)	PE		M-MLV RT	nCas9^H840A	NGG	1-50			12种碱基替换插入(<15 bp) 缺失(<40 bp)	不受PAM的距离限制	Anzalone et al., 2019
	PPE		CaMV RT Retron RT							在植物中实现PE 应用	Lin et al., 2020
	pPE2		M-MLV RT							在植物中实现PE 应用	Xu et al., 2020a
	ePPE		M-MLV RT^{∆RNase H} M-MLV RT:NC			1-92			12种碱基替换插入(<40 bp) 缺失(<100 bp)	提高了在植物中的编辑效率	Zong et al., 2022
多重编辑	SWISSs		rAPOBEC1 ecTadA:ecTadA^*	nCas9^D10A	NG	C₃-C₁₆ A₄-A₇			C:G>T:A A:T>G:C Indels	具有A>G、C>T和Indels三重编辑功能	Li et al., 2020b
片段删除编辑	AFIDs		hAPOBEC3A hAPOBEC3Bctd	Cas9	NGG	C₁-C₁₄			多核苷酸删除	精准高效、可预测的多核苷酸缺失	Wang et al., 2020

表1 各类碱基编辑器的特点

Table 1 Characteristics of all kinds of base editors

编辑技术	编辑器	脱氨酶/逆转录酶		Cas蛋白	PAM (5′-3′)		活性窗口	编辑类型		特点	参考文献
胞嘧啶碱基编辑(CBEs)	BE3	rAPOBEC1		nCas9^D10A	NGG		C₄-C₈	C:G>T:A		实现C>T替换, 无需DSB或提供模板	Komor et al., 2016
	nCas9-PBE	rAPOBEC1		nCas9^D10A	NGG		C₃-C₉			在植物中实现精准高效的C>T替换	Zong et al., 2017
	Target-AID	PmCDA1		nCas9^D10A	NGG		C₁-C₅			具有较好的GC碱基编辑能力	Nishida et al., 2016
	dCas9-AIDx	hAID		dCas9	NCN		C₄-C₉			具有较好的GC碱基编辑能力	Ma et al., 2016
	A3A^Y130F- CBE_V01 A3A^Y130F- CBE_V04	hAPOBEC3A^Y130F		nCas9^D10A	NGG		C₄-C₁₅			活性窗口拓宽至12 nt	Ren et al., 2021a
	eCDAL	LjCDA1L-4		nCas9^D10A	NGG		C₁-C₁₂			活性窗口拓宽至12 nt	Xu et al., 2021
	BE3-PAPAPAP	rAPOBEC1		nCas9^D10A	NGG		C₅-C₆			活性窗口缩小至1-2 nt	Tan et al., 2019
	PhieCBEs	evorAPOCBEC1 evoFERNY evoCDA1 hA3A		nCas9-NG eCas9n- NG	NG NGG		C_-1-C₁₅			编辑窗口广	Zeng et al., 2020
	BE3^R126E BE3^R132E YE1-BE3	rAPOBEC1^R126E rAPOBEC1^R132E rAPOBEC1^W90Y+R126E		nCas9^D10A	NGG		C₅-C₇			脱靶率显著降低	Zuo et al., 2020
腺嘌呤碱基编辑(ABEs)	ABE7.10	ecTadA:ecTadA^*		nCas9^D10A	NGG		A₄-A₇	A:T>G:C		实现A>G替换, 无需DSB或提供模板	Gaudelli et al., 2017
	PABE	ecTadA:ecTadA^*		nCas9^D10A	NGG		A₄-A₈			在植物中实现精准高效的A>G替换	Li et al., 2018b
	ABE-Ps	ecTadA:ecTadA^*		nCas9^D10A SaCas9	NGG NNNRRT		A₁-A₁₅			在植物中实现精准高效的A>G替换	Hua et al., 2018
	rBE14	ecTadA:ecTadA^*		nCas9^D10A	NGG		A₅-A₇			荧光检测编辑植株	Yan et al., 2018
	rABE8e	TadA8e^V106W		nCas9^D10A nCas9-NG	NG NGG		A₅-A₆			极高的靶点编辑效率和碱基纯合替换效率	Wei et al., 2021
	PhieABEs	TadA8e		nCas9-NG SpGn SpRYn	NG NGN NNN		A₁-A₁₄			编辑效率高, 几近无PAM靶向, 窗口广	Tan et al., 2022
	SpRY-ABE8e	ecTadA:ecTadA^*		nCas9^D10A	NNN		A₃-A₁₀			几近无PAM靶向	Ren et al., 2021b
	ABE7.10^F148A	ecTadA^F148A: ecTadA^*F148A		nCas9^D10A	NGG		A₅-A₆			活性窗口缩小至1-2 nt	Zhou et al., 2019
CG碱基编辑(GBEs)	GBE	AID rAPOBEC1		nCas9^D10A	NGG		C₃-C₇	C:G>A:T/G:C		实现嘧啶与嘌呤间的颠换	Zhao et al., 2021
CG碱基编辑(GBEs)	CGBE	rAPOBEC1		nCas9^D10A	NGG		C₅-C₆	C:G>A:T/G:C		实现嘧啶与嘌呤间的颠换	Chen et al., 2021
双碱基编辑(A&CBE)	A&C-BEmax	hAID ecTadA:ecTadA^*		nCas9^D10A	NGG		C₂-C₁₇ A₄-A₇	C:G>T:A A:T>G:C		实现A、C共编辑	Zhang et al., 2020
	Target-ACE	PmCDA1 ecTadA:ecTadA^*			NGG		C₁-C₁₀ A₄-A₈				Sakata et al., 2019
	SPACE	PmCDA1 ecTadA^*			NGG		C₂-C₇ A₄-A₇				Grünewald et al., 2020
	ACBE	PmCDA1 ecTadA:ecTadA^*			NGG		C₁-C₇ A₄-A₆				Xie et al., 2020
	STEME	hAPOBEC3A ecTadA:ecTadA^*			NG NGD		C₁-C₁₇ A₄-A₈			在植物中实现A、 C共编辑	Li et al., 2020a
	pDuBE1	TadA8e			NGG		C₁-C₁₀ A₂-A₉			在植物中实现A、 C共编辑	Xu et al., 2021
编辑技术	编辑器		脱氨酶/逆转录酶	Cas蛋白	PAM (5′-3′)	活性窗口			编辑类型	特点	参考文献
先导编辑(PEs)	PE		M-MLV RT	nCas9^H840A	NGG	1-50			12种碱基替换插入(<15 bp) 缺失(<40 bp)	不受PAM的距离限制	Anzalone et al., 2019
	PPE		CaMV RT Retron RT							在植物中实现PE 应用	Lin et al., 2020
	pPE2		M-MLV RT							在植物中实现PE 应用	Xu et al., 2020a
	ePPE		M-MLV RT^{∆RNase H} M-MLV RT:NC			1-92			12种碱基替换插入(<40 bp) 缺失(<100 bp)	提高了在植物中的编辑效率	Zong et al., 2022
多重编辑	SWISSs		rAPOBEC1 ecTadA:ecTadA^*	nCas9^D10A	NG	C₃-C₁₆ A₄-A₇			C:G>T:A A:T>G:C Indels	具有A>G、C>T和Indels三重编辑功能	Li et al., 2020b
片段删除编辑	AFIDs		hAPOBEC3A hAPOBEC3Bctd	Cas9	NGG	C₁-C₁₄			多核苷酸删除	精准高效、可预测的多核苷酸缺失	Wang et al., 2020

图1 单(CBE、ABE、GBE)、双(A&C-BEmax)碱基编辑器工作原理 (A) BE3介导的C>T替换(在nCas9D10A (蓝色)、胞苷脱氨酶rAPOBEC1 (红色)和尿嘧啶糖基化酶抑制剂(UGI) (绿色)的作用下, 系统BE3将活性窗口中的C脱氨成U, 诱导细胞启动DNA修复实现C到T的替换; 红色三角形表示切口处); (B) ABE7.10介导的A>G替换(在腺苷脱氨酶ecTadA:ecTadA* (黄色和橙色)的作用下, 系统ABE7.10将活性窗口中的A脱氨成I, 并在DNA修复后实现A到G的替换); (C) A&C-BEmax介导的C>T与A>G共替换(在胞嘧啶脱氨酶hAID (红色)、ecTadA:ecTadA*和2个UGI的作用下, 系统A&C-BEmax诱导DNA修复, 实现C到T、A到G的同时替换); (D) GBE介导的C>G颠换(在尿嘧啶-N-糖基化酶(UNG) (深蓝)的作用下, UNG将C脱氨生成的U水解为AP位点, 并在DNA修复后实现C到G的颠换)。PAM: 原间隔序列邻近基序

Figure 1 Technical principles of single (CBE, ABE, GBE) and dual (A&C-BEmax) base editors (A) BE3 mediated C to T base editing (mediated by nCas9D10A (blue), cytidine deaminase rAPOBEC1 (red) and uracil glycosylase inhibitor (UGI) (green), BE3 deaminates C in the active window into U and induces cells to start DNA repair and achieve the replacement of C to T; red triangle represents the notch); (B) ABE7.10 mediated A to G base editing (mediated by adenosine decease ecTadA:ecTadA* (yellow and orange), ABE7.10 deaminates A in the active window into I, and achieves A to G mutation after DNA repair); (C) A&C-BEmax mediated C to T and A to G base editing (mediated by cytidine deaminase hAID (red), ecTadA:ecTadA* and two UGI, A&C-BEmax induces DNA repair and achieves the simultaneous replacement of C to T and A to G); (D) GBE mediated C to G base editing (mediated by uracil-N-glycosylase (UNG) (deep blue), UNG hydrolyzes the U produced by C deamination to AP site, and achieves the transversion from C to G after DNA repair). PAM: Protospacer adjacent motif

图2 先导编辑器(PE)的工作原理在nCas9H840A (绿)和逆转录酶M-MLV (黄)的作用下, 系统PE将碱基序列连接到靶位点, 借助DNA修复实现任意碱基的转换以及小片段的插入或删除。PAM: 原间隔序列邻近基序; PBS: 引物结合位点

Figure 2 Technical principles of prime editor (PE) Mediated by nCas9H840A (green) and reverse transcriptase M-MLV (yellow), PE installs the base sequence into the target site, and achieves arbitrary base conversion and precise insertion and deletion of small fragments after DNA repair. PAM: Protospacer adjacent motif; PBS: Primer binding site

图3 多重编辑系统(SWISS)的工作原理 (A), (B) SWISS系统使用不同的scRNAs (MS2和boxB), 招募融合了相应蛋白(MCP和N22p)的rAPOBEC1或ecTadA:ecTadA*, 实现同时在不同位点的胞嘧啶碱基编辑(CBE)和腺嘌呤碱基编辑(ABE); (C) 使用1对sgRNA在第3个靶点产生DNA双链断裂(DSB), 诱导细胞进行同源定向修复(HDR), 产生随机突变。PAM: 原间隔序列邻近基序; UGI: 尿嘧啶糖基化酶抑制剂

Figure 3 Technical principles of simultaneous and wide-editing induced by a single system (SWISS) (A), (B) Mediated by different scRNAs (MS2 and boxB), SWISS recruits rAPOBEC1 or ecTadA:ecTadA* that combines the corresponding proteins (MCP and N22p) to achieve cytidine (CBE) and adenine (ABE) base editing at different sites; (C) Mediated by a pair of sgRNAs, Cas9 produces double strand break (DSB) at the third target, induces homology-directed repair (HDR) and produces random mutation. PAM: Protospacer adjacent motif; UGI: Uracil glycosylase inhibitor

图4 多核苷酸靶向删除系统(AFID)的工作原理 Cas9切割双链产生双链断裂(DSB), 尿嘧啶-DNA-糖基化酶(UDG)将C脱氨生成的U水解为AP位点, 并借助AP裂合酶(橙红色)和核酸外切酶(棕色), 实现靶点C到DSB切口之间的多核苷酸删除。PAM: 原间隔序列邻近基序

Figure 4 Technical principles of APOBEC-Cas9 fusion-induced deletion systems (AFIDs) Cas9 cuts both strands to form adouble strand break (DSB), uracil-DNA-glycosylase (UDG) hydrolyzes the U produced by C deamination to AP site, and achieve the polynucleotide deletion between target C and DSB with the help of AP lyase (orange red) and exonuclease (brown). PAM: Protospacer adjacent motif

图5 CRISPR干扰系统的工作原理 (A) dCas9阻止RNA聚合酶(RNAP)结合基因启动子, sgRNA介导dCas9结合目的基因启动子, 使RNAP (红色)无法与该基因启动子结合并进行转录; (B) dCas9阻断RNAP的转录延伸, sgRNA介导dCas9结合目的基因开放阅读框(ORF), 使RNAP无法继续转录延伸; (C) 转录抑制子阻止RNAP结合基因启动子, dCas9与转录抑制子(灰色)融合, 抑制子会阻止RNAP与目的基因启动子的结合; (D) 阻遏结合域(RBD)阻止目的基因的转录激活, dCas9与RBD (棕色)融合, RBD阻断转录因子(TFs)与目的基因的结合, 并与TFs结合形成强阻遏物抑制基因的转录表达。PAM: 原间隔序列邻近基序

Figure 5 Technical principles of CRISPR interference system (A) dCas9 prevents RNA polymerase (RNAP) from binding to gene promoter, sgRNA mediated dCas9 binding to the target gene promoter, so that RNAP (red) can not bind to the gene promoter to start transcribing; (B) dCas9 blocks the transcriptional extension of RNAP, sgRNA mediates dCas9 binding to the target gene open reading frame (ORF), making RNAP unable to continue transcriptional extension; (C) Transcriptional repressors prevent RNAP from binding to gene promoters, dCas9 fuses with the transcription inhibitor (gray), which prevents the binding of RNAP to the promoter of the target gene; (D) The repressor binding domain (RBD) blocks the transcriptional activation of the target gene, dCas9 fuses with the RBD (brown), RBD blocked the binding of transcription factors (TFs) to the target gene and combined with TFs to form a strong repressor to inhibit the transcription of the gene. PAM: Protospacer adjacent motif

图6 CRISPR激活系统的工作原理 (A) 转录激活子激活目的基因转录, dCas9与转录激活子(洋红色)融合, 招募RNA聚合酶(RNAP)并激活目的基因的转录; (B)-(E) 不同CRISPRa系统示意图, 包括TALs (橙红色)、VP64 (绿色)、MS2 (浅黄色)、MCP (深黄色)、GCN4 (湛蓝色)和scFv (明黄色)。PAM: 原间隔序列邻近基序

Figure 6 Technical principles of CRISPR activation system (A) Transcriptional activator activates the transcription of the target gene, dCas9 fuses with transcription activator (magenta) to recruit RNA polymerase (RNAP) and activates the transcription of the target gene; (B)-(E) Schematic diagram of different CRISPRa systems, including TALs (orange red), VP64 (green), MS2 (light yellow), MCP (deep yellow), GCN4 (azure blue) and scFv (bright yellow). PAM: Protospacer adjacent motif

图7 基因组修饰系统去甲基化和甲基化的工作原理 (A) DNA去甲基化修饰, dCas9-SunTag系统招募人脱甲基酶TET1cd (棕色), 在目的基因的启动子区使DNA去甲基化, Me表示甲基化; (B) DNA甲基化修饰, dCas9-SunTag系统招募烟草DRM甲基转移酶催化结构域NtDRMcd (绿色), 在目的基因启动子区使DNA甲基化。PAM: 原间隔序列邻近基序

Figure 7 Technical principles of demethylation and methylation of genome modification systems (A) DNA demethylation modification, dCas9-SunTag system recruits human demethylase TET1cd (brown) to demethylate DNA in the promoter region of the target gene, Me represents methylation; (B) DNA methylation modification, dCas9-SunTag system recruits tobacco DRM methyltransferase catalytic domain NtDRMcd (green) to methylate DNA in the promoter region of the target gene. PAM: Protospacer adjacent motif

表2 碱基编辑器在作物中的应用

Table 2 Application of base editors in crop

物种		品种	靶基因	转化方法	编辑技术	脱氨酶/逆转录酶		编辑类型	编辑窗口	编辑效率(%)		参考文献
水稻(Oryza sativa)		中花11	CDC48 NRT1.1B-T1	农杆菌AGL1	CBEs	hAPOBEC3A		C:G>T:A	C₂-C₁₆	44.1- 82.9		Zong et al., 2018
		日本晴	CDC48	农杆菌AGL1		rAPOBEC1		C:G>T:A	C₃-C₈	43.48		Zong et al., 2017
		Kitaake	Pi-d2 FLS2	农杆菌EHA105		hAID^*Δ		C:G>T:A	C₃-C₇	30.8-57		Ren et al., 2018
		中花11	ACC-T1 ALS-T1 CDC48-T3 DEP1-T1/2 NRT1.1B-T1	农杆菌AGL1	ABEs	ecTadA: ecTadA^*		A:T>G:C	A₄-A₈	15.8- 59.1		Li et al., 2018b
		日本晴	SPL14/16/17/18 SLR1	农杆菌EHA105		ecTadA: ecTadA^*		A:T>G:C	A₅-A₁₄	12.5- 61.3		Hua et al., 2018
		Kitaake	SERK2 WRKY45	农杆菌EHA105		ecTadA: ecTadA^*		A:T>G:C	A₄-A₇	32.05- 62.26		Yan et al., 2018
		中花11	ACC	农杆菌AGL1	STEMEs	hAPOBEC3A ecTadA: ecTadA^*		C:G>T:A; A:T>G:C	C₁-C₁₇ A₄-A₈	3.84		Li et al., 2020a
		中花11	CDC48-T1 ALS-T2	农杆菌EHA105	PEs	M-MLV		G:C>T:A 插入(≤3 bp) 删除(≤6 bp)	N₁-N₆	2.6-21.8		Lin et al., 2020
		日本晴	ALS-1/2 ACC-1 DEP1	农杆菌EHA105	PEs	M-MLV		C:G>T:A A:T>G:C G:C>T:A G:C>C:G G:C>A:T T:A>A:T A:T>C:G	N₁₈-N₃₃	1.7-26		Xu et al., 2020b
物种	品种		靶基因	转化方法	编辑技术	脱氨酶/逆转录酶	编辑类型		编辑窗口	编辑效率(%)	参考文献
	中花11		ALS-T2 ACC-T2 BADH-indels	农杆菌AGL1	SWISS	hAPOBEC3A ecTadA: ecTadA^*	C:G>T:A A:T>G:C C:G>G:C 删除(≤45 bp)		C₆-C₇ A₄-A₇	7.3	Li et al., 2020b
	中花11		CDC48-T2 SPL14 SWEET14	农杆菌AGL1	AFID	hAPOBEC3A	删除(≤16 bp)		C₂-C₁₇	22.2- 55.8	Wang et al., 2020
小麦(Triticum aestivum)	Kenong 199		ALS MTL	基因枪	CBEs	hAPOBEC3A	C:G>T:A		C_-9-C₁₃	16.7- 22.5	Zong et al., 2018
	Bobwhite		LOX2-S1	基因枪	CBEs	rAPOBEC1	C:G>T:A		C₃-C₉	1.25	Zong et al., 2017
	Kenong 199		ALS	基因枪	CBEs	rAPOBEC1	C:G>T:A		C_-1-C₇	22-78	Zhang et al., 2019
	Kenong 199		DEP1 GW2	基因枪	ABEs	ecTadA: ecTadA^*	A:T>G:C		A₅-A₈	0.4-1.1	Li et al., 2018a
	Kenong 199		miR396 GASR6	基因枪	AFID	hAPOBEC3A	删除(≤35 bp)		C_-12-C₂₃	25-37.5	Wang et al., 2020
番茄(Solanum lycopersicum)	WVA106		ALS1	农杆菌C58 pGV2260	CBEs	PmCDA1	C:G>T:A		C₇	34.7	Veillet et al., 2019
	WVA106		ALS1	农杆菌C58 pGV2260	CBEs	PmCDA1	C:G>T:A		C₁-C₈	20.59	Veillet et al., 2020
	Micro- Tom		DELLA	农杆菌	CBEs	PmCDA1	C:G>T:A		C₁-C₃	50.5	Shimatani et al., 2017
马铃薯 (S. tuberosum)	Désirée		GBSS1 DMR6-1	PEG	CBEs	hAPOBEC3A	C:G>T:A		C₃-C₁₀	8-16.67	Veillet et al., 2020
	Désirée		GBSS-T6	PEG	CBEs	hAPOBEC3A	C:G>T:A		C₁-C₁₃	6.5	Jiang et al., 2022
	Désirée		ALS1	农杆菌C58 pGV2260	CBEs	PmCDA1	C:G>T:A		C₁-C₈	25	Veillet et al., 2019
大豆 (Glycine max)	Jack		FT2a FT4	农杆菌	CBEs	rAPOBEC1	C:G>T:A		C₆-C₇	5.88- 18.2	Cai et al., 2020
棉花(Gossypium hirsutum)	Jin668		CLA PEBP	农杆菌GV3101	CBEs	rAPOBEC1	C:G>T:A		C₄-C₈	26.67- 57.78	Qin et al., 2020
玉米 (Zea mays)	Zong31		CENH3	农杆菌AGL1	CBEs	rAPOBEC1	C:G>T:A		C₃-C₈	10.1	Zong et al., 2017
玉米 (Zea mays)	ND73		ALS1/2	农杆菌LBA4404/ pVS1-VIR2	PEs	CmYLCV	C:G>T:A A:T>C:G T:A>G:C T:A>C:G C:G>A:T G:C>T:A G:C>A:T G:C>C:G		N₃-N₄₆	6.5-53.2	Jiang et al., 2020
油菜(Brassica napus)	J9712		ALS1	农杆菌	CBEs	rAPOBEC1	C:G>T:A		C₅-C₇	1.8	Wu et al., 2020
西瓜(Citrullus lanatus)	ZG94		ALS	农杆菌EHA105	CBEs	rAPOBEC1	C:G>T:A		C₇-C₈	23	Tian et al., 2018

表2 碱基编辑器在作物中的应用

Table 2 Application of base editors in crop

物种		品种	靶基因	转化方法	编辑技术	脱氨酶/逆转录酶		编辑类型	编辑窗口	编辑效率(%)		参考文献
水稻(Oryza sativa)		中花11	CDC48 NRT1.1B-T1	农杆菌AGL1	CBEs	hAPOBEC3A		C:G>T:A	C₂-C₁₆	44.1- 82.9		Zong et al., 2018
		日本晴	CDC48	农杆菌AGL1		rAPOBEC1		C:G>T:A	C₃-C₈	43.48		Zong et al., 2017
		Kitaake	Pi-d2 FLS2	农杆菌EHA105		hAID^*Δ		C:G>T:A	C₃-C₇	30.8-57		Ren et al., 2018
		中花11	ACC-T1 ALS-T1 CDC48-T3 DEP1-T1/2 NRT1.1B-T1	农杆菌AGL1	ABEs	ecTadA: ecTadA^*		A:T>G:C	A₄-A₈	15.8- 59.1		Li et al., 2018b
		日本晴	SPL14/16/17/18 SLR1	农杆菌EHA105		ecTadA: ecTadA^*		A:T>G:C	A₅-A₁₄	12.5- 61.3		Hua et al., 2018
		Kitaake	SERK2 WRKY45	农杆菌EHA105		ecTadA: ecTadA^*		A:T>G:C	A₄-A₇	32.05- 62.26		Yan et al., 2018
		中花11	ACC	农杆菌AGL1	STEMEs	hAPOBEC3A ecTadA: ecTadA^*		C:G>T:A; A:T>G:C	C₁-C₁₇ A₄-A₈	3.84		Li et al., 2020a
		中花11	CDC48-T1 ALS-T2	农杆菌EHA105	PEs	M-MLV		G:C>T:A 插入(≤3 bp) 删除(≤6 bp)	N₁-N₆	2.6-21.8		Lin et al., 2020
		日本晴	ALS-1/2 ACC-1 DEP1	农杆菌EHA105	PEs	M-MLV		C:G>T:A A:T>G:C G:C>T:A G:C>C:G G:C>A:T T:A>A:T A:T>C:G	N₁₈-N₃₃	1.7-26		Xu et al., 2020b
物种	品种		靶基因	转化方法	编辑技术	脱氨酶/逆转录酶	编辑类型		编辑窗口	编辑效率(%)	参考文献
	中花11		ALS-T2 ACC-T2 BADH-indels	农杆菌AGL1	SWISS	hAPOBEC3A ecTadA: ecTadA^*	C:G>T:A A:T>G:C C:G>G:C 删除(≤45 bp)		C₆-C₇ A₄-A₇	7.3	Li et al., 2020b
	中花11		CDC48-T2 SPL14 SWEET14	农杆菌AGL1	AFID	hAPOBEC3A	删除(≤16 bp)		C₂-C₁₇	22.2- 55.8	Wang et al., 2020
小麦(Triticum aestivum)	Kenong 199		ALS MTL	基因枪	CBEs	hAPOBEC3A	C:G>T:A		C_-9-C₁₃	16.7- 22.5	Zong et al., 2018
	Bobwhite		LOX2-S1	基因枪	CBEs	rAPOBEC1	C:G>T:A		C₃-C₉	1.25	Zong et al., 2017
	Kenong 199		ALS	基因枪	CBEs	rAPOBEC1	C:G>T:A		C_-1-C₇	22-78	Zhang et al., 2019
	Kenong 199		DEP1 GW2	基因枪	ABEs	ecTadA: ecTadA^*	A:T>G:C		A₅-A₈	0.4-1.1	Li et al., 2018a
	Kenong 199		miR396 GASR6	基因枪	AFID	hAPOBEC3A	删除(≤35 bp)		C_-12-C₂₃	25-37.5	Wang et al., 2020
番茄(Solanum lycopersicum)	WVA106		ALS1	农杆菌C58 pGV2260	CBEs	PmCDA1	C:G>T:A		C₇	34.7	Veillet et al., 2019
	WVA106		ALS1	农杆菌C58 pGV2260	CBEs	PmCDA1	C:G>T:A		C₁-C₈	20.59	Veillet et al., 2020
	Micro- Tom		DELLA	农杆菌	CBEs	PmCDA1	C:G>T:A		C₁-C₃	50.5	Shimatani et al., 2017
马铃薯 (S. tuberosum)	Désirée		GBSS1 DMR6-1	PEG	CBEs	hAPOBEC3A	C:G>T:A		C₃-C₁₀	8-16.67	Veillet et al., 2020
	Désirée		GBSS-T6	PEG	CBEs	hAPOBEC3A	C:G>T:A		C₁-C₁₃	6.5	Jiang et al., 2022
	Désirée		ALS1	农杆菌C58 pGV2260	CBEs	PmCDA1	C:G>T:A		C₁-C₈	25	Veillet et al., 2019
大豆 (Glycine max)	Jack		FT2a FT4	农杆菌	CBEs	rAPOBEC1	C:G>T:A		C₆-C₇	5.88- 18.2	Cai et al., 2020
棉花(Gossypium hirsutum)	Jin668		CLA PEBP	农杆菌GV3101	CBEs	rAPOBEC1	C:G>T:A		C₄-C₈	26.67- 57.78	Qin et al., 2020
玉米 (Zea mays)	Zong31		CENH3	农杆菌AGL1	CBEs	rAPOBEC1	C:G>T:A		C₃-C₈	10.1	Zong et al., 2017
玉米 (Zea mays)	ND73		ALS1/2	农杆菌LBA4404/ pVS1-VIR2	PEs	CmYLCV	C:G>T:A A:T>C:G T:A>G:C T:A>C:G C:G>A:T G:C>T:A G:C>A:T G:C>C:G		N₃-N₄₆	6.5-53.2	Jiang et al., 2020
油菜(Brassica napus)	J9712		ALS1	农杆菌	CBEs	rAPOBEC1	C:G>T:A		C₅-C₇	1.8	Wu et al., 2020
西瓜(Citrullus lanatus)	ZG94		ALS	农杆菌EHA105	CBEs	rAPOBEC1	C:G>T:A		C₇-C₈	23	Tian et al., 2018

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基于CRISPR/Cas9的基因编辑技术研究进展及其在植物中的应用

Advance in Gene-editing Technology Based on CRISPR/Cas9 and Its Application in Plants

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