Plant Prime Editing: A New Direction in Crop Breeding

doi:10.16819/j.1001-7216.2025.241005

Abstract

Abstract:

Plant Prime Editing (PPE) provides a novel approach for precise genome modification in plants, overcoming the limitations of conventional gene editing methods that rely on double-strand breaks and exogenous donor DNA. It enables not only arbitrary base substitutions, but also precise insertions and deletions of small to large DNA fragments, with reduced off-target effects. This review systematically summarizes the latest research advances in PPE technology globally, including its development history and working mechanism, applications in crop breeding and trait improvement, and representative cases. It also discusses the expansion of prime editing into large-scale genome editing and multiplex gene editing. To address the bottlenecks faced by PPE systems in plant genetic transformation, several potential solutions are proposed. Finally, the broad application prospects of PPE technology in plant genetic improvement and its potential integration with artificial intelligence (AI) in future research are outlined.

Key words: plant prime editing, CRISPR/Cas, precision gene editing, crop breeding, plant genetic transformation

摘要：

植物引导编辑技术(Plant Prime Editing，PPE)为植物基因组的精准改良提供了全新途径，克服了传统基因编辑方法中依赖双链断裂和外源供体DNA的局限性，不仅可以实现任意类型碱基替换，还可以进行小片段乃至大范围片段的精准插入和删除，并且脱靶率较低。本文详细阐述了PPE技术在国内外的最新研究进展，包括其发展历程与工作原理、在作物育种与性状改良中的应用，及PE技术在大规模基因组编辑和多重基因编辑等领域的拓展。针对PPE系统在植物遗传转化过程中遇到的瓶颈问题，提出了若干解决方案，并展望了PPE技术在植物遗传改良中的广泛应用前景及未来与人工智能(Artificial Intelligence，AI)相结合的研究方向。

关键词: 植物引导编辑技术, CRISPR/Cas, 精准编辑, 作物育种, 植物遗传转化

SONG Anqi, WU Songquan, MA Qiuyue, BAN Wanning, LIU Xiangguo, JIN Yongmei. Plant Prime Editing: A New Direction in Crop Breeding[J]. Chinese Journal OF Rice Science, 2025, 39(6): 711-730.

宋安琪, 吴松权, 马秋月, 班宛宁, 刘相国, 金永梅. 植物引导编辑技术——作物育种的新方向[J]. 中国水稻科学, 2025, 39(6): 711-730.

Figures/Tables 6

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系统名称	系统组件			PAM	启动子	编辑效率 (%)	物种	参考文献
系统名称	Cas9 Nickcase	逆转录酶	pegRNA	PAM	启动子	编辑效率 (%)	物种	参考文献
PPE2	nCas9(H840A) 密码子优化	M-MLV RT密码子优化(D200N/L603W/ T330P/T306K/W31S)	原始的pegRNA	NGG	OsU3/TaU6, TaU3	0~21.8 (Pr, Re)^*	水稻, 小麦	[34]
PPE3			pegRNA+nicking sgRNA
PPE3b			Nicking sgRNA中的spacer序列与被编辑后的序列互补
PPE-CaMV		CaMV RT	原始的pegRNA	NGG		5.8、0.3 (Pr, Re)	水稻
PPE-ribozyme	nCas9(H840A) proteintranscript +polymerase II (Pol II)	Retron RT	Ribozyme-processed pegRNA	NGG		>9 (Pr, Re)	水稻
PPE3-V01, PPE3b-V01	nCas9(H840A) 密码子优化	M-MLV RT密码子优化	pegRNA+nicking sgRNA	NGG	OsU3, OsU6	0.05~0.4 (Pr)	水稻	[35]
PPE2-V02, PPE3-V02	nCas9(H840A) 密码子优化	M-MLV RT密码子优化、核定位信号配置优化	pegRNA	NGG	OsU6	<1.55 (Pr)	水稻	[35]
Prime editor basic (PPE3)	nCas9(H840A) C端+核定位信号NLS	在M-MLV RT上引入了 6个点突变(H9Y/ D200N/T306K/W313F/ T330P/L603W)	使用多顺反子tRNA策略同时产生pegRNA和nicking sgRNA	NGG	Actin	2.22~9.38 (Pr)	水稻	[36]
PE-P1	nCas9(H840A) 密码子优化	M-MLV RT密码子优化、核定位信号配置优化、 Link序列类型优化	原始的pegRNA	NGG	OsU3, OsU6a	0%~1.4 (Re)	水稻	[37]
PE-P2	nCas9(H840A) 密码子优化	在PE-P1的基础上，M- MLV 的C端添加了多顺反子连接子：2A self-cleaving peptides(P2A)	pegRNA+由增强的esgRNA组成的pegRNA		OsU3, OsU6a	1.7~26 (Re)	水稻	[37]
pPE2	nCas9(H840A) 密码子优化	M-MLV RT密码子优化(D200N/L603W/ T330P/T306K/ W313F)、 Link序列类型优化	原始的pegRNA	NGG	OsU3	0~31.3 (Re)	水稻	[38]
pPE3, pPE3b			pegRNA+nicking sgRNA		TaU3
Surrogate pPE2			采用双pegRNA策略，“编辑HPT起始密码子pegRNA+带有编辑信息pegRNA”	NGG	OsU3
Sp-PE2	nCas9(H840A) 密码子优化	M-MLV RT密码子优化(D200N/L603W/T306K/ W313F/T330P)、核定位信号配置优化	原始的pegRNA	NGG	OsU6	15.60 (Re)	水稻	[39]

系统名称	系统组件			PAM	启动子	编辑效率 (%)	物种	参考文献
系统名称	Cas9 Nickcase	逆转录酶	pegRNA	PAM	启动子	编辑效率 (%)	物种	参考文献
PPE2	nCas9(H840A) 密码子优化	M-MLV RT密码子优化(D200N/L603W/ T330P/T306K/W31S)	原始的pegRNA	NGG	OsU3/TaU6, TaU3	0~21.8 (Pr, Re)^*	水稻, 小麦	[34]
PPE3			pegRNA+nicking sgRNA
PPE3b			Nicking sgRNA中的spacer序列与被编辑后的序列互补
PPE-CaMV		CaMV RT	原始的pegRNA	NGG		5.8、0.3 (Pr, Re)	水稻
PPE-ribozyme	nCas9(H840A) proteintranscript +polymerase II (Pol II)	Retron RT	Ribozyme-processed pegRNA	NGG		>9 (Pr, Re)	水稻
PPE3-V01, PPE3b-V01	nCas9(H840A) 密码子优化	M-MLV RT密码子优化	pegRNA+nicking sgRNA	NGG	OsU3, OsU6	0.05~0.4 (Pr)	水稻	[35]
PPE2-V02, PPE3-V02	nCas9(H840A) 密码子优化	M-MLV RT密码子优化、核定位信号配置优化	pegRNA	NGG	OsU6	<1.55 (Pr)	水稻	[35]
Prime editor basic (PPE3)	nCas9(H840A) C端+核定位信号NLS	在M-MLV RT上引入了 6个点突变(H9Y/ D200N/T306K/W313F/ T330P/L603W)	使用多顺反子tRNA策略同时产生pegRNA和nicking sgRNA	NGG	Actin	2.22~9.38 (Pr)	水稻	[36]
PE-P1	nCas9(H840A) 密码子优化	M-MLV RT密码子优化、核定位信号配置优化、 Link序列类型优化	原始的pegRNA	NGG	OsU3, OsU6a	0%~1.4 (Re)	水稻	[37]
PE-P2	nCas9(H840A) 密码子优化	在PE-P1的基础上，M- MLV 的C端添加了多顺反子连接子：2A self-cleaving peptides(P2A)	pegRNA+由增强的esgRNA组成的pegRNA		OsU3, OsU6a	1.7~26 (Re)	水稻	[37]
pPE2	nCas9(H840A) 密码子优化	M-MLV RT密码子优化(D200N/L603W/ T330P/T306K/ W313F)、 Link序列类型优化	原始的pegRNA	NGG	OsU3	0~31.3 (Re)	水稻	[38]
pPE3, pPE3b			pegRNA+nicking sgRNA		TaU3
Surrogate pPE2			采用双pegRNA策略，“编辑HPT起始密码子pegRNA+带有编辑信息pegRNA”	NGG	OsU3
Sp-PE2	nCas9(H840A) 密码子优化	M-MLV RT密码子优化(D200N/L603W/T306K/ W313F/T330P)、核定位信号配置优化	原始的pegRNA	NGG	OsU6	15.60 (Re)	水稻	[39]

系统名称	系统组件						编辑类型			编辑效率 (%)		物种		参考文献
系统名称	Cas9 Nickcase		逆转录酶		pegRNA		编辑类型			编辑效率 (%)		物种		参考文献
MS2PE	nCas9(H840A)密码子优化		M-MLV密码子优化(D200N/L603W/ T330P/T306K/W31S)与MS2 RNA 结合蛋白基因MCP融合		在原始pegRNA的3'端添加MS2 RNA结合蛋白基因MCP		3-bp subs			1.7~55.6 (Pr, Re)		水稻		[41]
PE-P2 (水稻)	nCas9(H840A)密码子优化		M-MLV依旧在nCas9的C端融合		pegRNA+增强的esgRNA		1-bp subs			0~5.9(Re)		水稻		[42]
PE-P3 (水稻)			M-MLV从nCas9 C端融合变为N 端融合		pegRNA+增强的esgRNA					2.6~92.3(Re)		水稻
PE-P2 (玉米)			M-MLV-02玉米密码子优化；依旧在nCas9 C端融合		Target+esgRNA和pegRNA					0~7.7(Pr)		玉米
PE-P3 (玉米)			M-MLV-02玉米密码子优化，从 nCas9 C端融合变为N端融合		Target+esgRNA和pegRNA					2.9~80(Pr)		玉米
ePPE	nCas9(H840A)密码子优化		在原始PPE基础上，去除RT的 RNase H结构域并添加病毒核衣壳(NC)蛋白		原始的pegRNA		1-2-bp subs；3-bp ins；2-bp del； 15-90-bp ins or del			0~31.5 (Re)		水稻		[43]
PPE3- evopreQ1	nCas9(H840A)密码子优化		M-MLV密码子优化(D200N/ L603W/T330P/T306K/W31S)		在PPE3基础上, 结构化pegRNA，在原始pegRNA的3'端添加8 bp linker和evopreQ1/ mpknot以提高稳定性		1-3-bp subs； 3-bp ins			2.6~60.5 (Pr, Re)		水稻		[44]
PPE3-mpknot	nCas9(H840A)密码子优化		M-MLV密码子优化(D200N/ L603W/T330P/T306K/W31S)				1-3-bp subs； 3-bp ins			0~6.3 (Pr, Re)		水稻		[44]
pPEmax	nCas9(R221K/N394K/H840A) 密码子优化		M-MLV密码子优化，N端添加 NLS-embedded linker，C端添加异质串联NLS		epegRNA：在pegRNA 3'端添加evopreQ1		1-bp ins； 1-2-bp subs			14.58~66.67 (Re)		水稻		[45]
pPEmax- MLHdn			在pPEmax的基础上，C端添加MLH1dn，以抑制MMR途径		epegRNA：在pegRNA 3'端添加evopreQ1					0.18~2.3 (Re)
enpPE2			同pPEmax		在U6复合启动子驱动下，构成tRNA(Gly-tRNA):: pegRNA::evopreQ1:: HDV表达盒					64.58~77(Re)
pINPE2	nCas9(H840A)密码子优化， N端添加小肽		M-MLV C端添加异质串联NLSs		epegRNA：在pegRNA 3'端添加evopreQ1					0.01~0.8 (Re)
phyPE2	nCas9(H840A)密码子优化		M-MLV N端添加DBD linker (hRad51-ssDBD)		epegRNA：在pegRNA 3'端添加evopreQ1					<0.1(Re)
ePE3max	nCas9(R221K/N394K/H840A) 密码子优化		M-MLV密码子优化(D200N/ L603W/T330P/T306K/W31S)		U6复合启动子驱动的表达盒中添加tRNA(Gly-tRNA)和HDV核酶，并添加nicking sgRNA		1-bp ins； 1-3-bp subs			~40(Re)		水稻		[46]
ePE5max	nCas9(R221K/N394K/H840A) 密码子优化		M-MLV C端添加MLH1dn，以抑制MMR途径		U6复合启动子驱动的表达盒中添加tRNA(Gly-tRNA)和HDV核酶，并添加nicking sgRNA		1-bp ins； 1-3-bp subs			~40(纯合率提高)(Re)		水稻		[46]
PrimeRoot	nCas9(H840A)密码子优化		M-MLV去除RNase H结构域，并添加重组酶		采用“双ePPE”策略，使用了两个相邻的epegRNA，每个模板都包含一个仅与另一个epegRNA模板具有同源性的RT模板		720-bp，1.4-kb，4.9-kb, 7.7-kb，11.1-kb ins			0~8.3 (Pr, Re)		水稻		[47]
ePPE*	nCas9(H840A)密码子优化		M-MLV密码子优化(D200N/ V223A/T306K/W313F/T330P/ L603W)，去除RNase H结构域并添加病毒核衣壳(NC)蛋白；优化核定位信号		epegRNA：在pegRNA 3’端添加evopreQ1		1-3-bp subs； 3-6-bp del； 4-bp ins			0~18.9 (Pr, Re)		小麦		[48]
ePPEmax	nCas9(H840A)密码子优化
ePPEmax*	nCas9(R221K/N394K/H840A) 密码子优化
ePPEplus	nCas9(R221K/N394K/H840A) 密码子优化
CMPE- ePPEplus	Csy4核糖核酸内切酶				串联多个(e)pegRNA		6- bp del; 1-bp subs			2~21.6; 19.6~86.3 (Pr, Re)
ePE5max (玉米)	nCas9(R221K/N394K/H840A) 密码子优化		M-MLV C端添加MLH1dn玉米同源物，以抑制MMR途径		U6复合启动子驱动的双pegRNA表达盒中添加tRNA(Gly-tRNA)和HDV核酶		1-bp subs			Homo: 1.4~3 hetero: 15~75 (Re)		玉米		[49]
pCXPE01, pCXPE02, pCXPE03	nCas9(H840A)密码子优化		M-MLV植物密码子优化		原始的pegRNA		2-bp subs； 1-3-bp del			0.025~1.66; 2.6 (Pr)		番茄		[50]
pPPED	nCas9(H840A)密码子优化		M-MLV植物密码子优linker序列类型优化		原始的pegRNA		2-bp subs			0.06 (Pr)		本氏烟草		[51]
系统名称		系统组件						编辑类型	编辑效率 (%)		物种		参考文献
系统名称		Cas9 Nickcase		逆转录酶		pegRNA		编辑类型	编辑效率 (%)		物种
pPPEDs								2-bp subs； 25-66-bp ins	0.07±0.12 (Re)		拟南芥
pPPEM								2-bp subs；25-66-bp ins	0.7~2.2 (Pr)		水稻
pAct-PPE		nCas9(H840A)密码子优化		M-MLV植物密码子优化		原始的pegRNA		3-bp subs	0~5 (Re)		小立碗藓、马铃薯		[52]
PPE2 PPE3 PPE3b		nCas9(H840A)密码子优化		M-MLV植物密码子优化		原始的pegRNA和nicking sgRNA		1-bp subs	0.2~0.5 (Pr)		花生，鹰嘴豆，豇豆		[53]
PE-Nt2		nCas9(H840A)密码子优化		M-MLV通过linker在nCas9 C 端融合		pegRNA与nesgRNA (用于切割非编辑链的增强sgRNA)通过tRNA序列连接		1-bp subs	0~1.4 (Re)		烟草		[54]
PE-Nt3				M-MLV通过linker在nCas9 N 端融合					1.1~7.5 (Re)
PE-Nt4				M-MLV通过linker在nCas9 N 端融合					1.3~16.3 (Re)
PE2 (v2)		nCas9(H840A)密码子优化		在nCas9-M-MLV 融合蛋白N端添加T5外切酶		原始的pegRNA、双pegRNA优化、 PBS的长度优化		4-bp ins； 4-bp del； 1−2-bp subs	3.5~48.65 (Pr, Re)		水稻		[55]
PE3-HS/AS/ DS		nCas9(H840A)密码子优化		M-MLV植物密码子优化；潮霉素代理系统、除草剂代理系统和潮霉素+除草剂双代理系统		tRNA-pegRNA(n)- tRNA-sgRNA(n)- tRNA		1-7-bp subs	1.3~54.2 (Re)		水稻		[56]
PE-DSM vector		nCas9(H840A)密码子优化		潮霉素+除草剂双代理多基因编辑系统		tRNA-pegRNA(n)- tRNA-sgRNA(n)- tRNA		1-7-bp subs	1.8~45.8 (Re)		水稻		[56]
DPE, TPE, QPE		nCas9(R221K/N394K/H840A)密码子优化		同ePE3max		pegRNA(n)-ngRNA(n)通过Gateway连接		2-3-bp subs, 28-bp ins	57.14 (Re)		水稻		[57]
-		nCas9(R221K/N394K/H840A) 密码子优化		同ePE5max		epegRNA：在pegRNA 3’端添加evopreQ1		4-bp ins	9.7 (Re)		番茄、拟南芥		[58]
GRAND		nCas9(H840A)密码子优化		M-MLV植物密码子优化		RTT部分对齐，但与双pegRNA中的目标序列非同源		46-bp subs	0.59~9.88 (Pr, Re)		水稻		[59]
PE5max		nCas9(R221K/N394K/H840A) 密码子优化		M-MLV C端添加MLH1dn，以抑制MMR途径		U6复合启动子驱动的双pegRNA表达盒中添加tRNA(Gly-tRNA)和HDV核酶		30-bp ins	47 (Re)		水稻		[60]

系统名称	系统组件						编辑类型			编辑效率 (%)		物种		参考文献
系统名称	Cas9 Nickcase		逆转录酶		pegRNA		编辑类型			编辑效率 (%)		物种		参考文献
MS2PE	nCas9(H840A)密码子优化		M-MLV密码子优化(D200N/L603W/ T330P/T306K/W31S)与MS2 RNA 结合蛋白基因MCP融合		在原始pegRNA的3'端添加MS2 RNA结合蛋白基因MCP		3-bp subs			1.7~55.6 (Pr, Re)		水稻		[41]
PE-P2 (水稻)	nCas9(H840A)密码子优化		M-MLV依旧在nCas9的C端融合		pegRNA+增强的esgRNA		1-bp subs			0~5.9(Re)		水稻		[42]
PE-P3 (水稻)			M-MLV从nCas9 C端融合变为N 端融合		pegRNA+增强的esgRNA					2.6~92.3(Re)		水稻
PE-P2 (玉米)			M-MLV-02玉米密码子优化；依旧在nCas9 C端融合		Target+esgRNA和pegRNA					0~7.7(Pr)		玉米
PE-P3 (玉米)			M-MLV-02玉米密码子优化，从 nCas9 C端融合变为N端融合		Target+esgRNA和pegRNA					2.9~80(Pr)		玉米
ePPE	nCas9(H840A)密码子优化		在原始PPE基础上，去除RT的 RNase H结构域并添加病毒核衣壳(NC)蛋白		原始的pegRNA		1-2-bp subs；3-bp ins；2-bp del； 15-90-bp ins or del			0~31.5 (Re)		水稻		[43]
PPE3- evopreQ1	nCas9(H840A)密码子优化		M-MLV密码子优化(D200N/ L603W/T330P/T306K/W31S)		在PPE3基础上, 结构化pegRNA，在原始pegRNA的3'端添加8 bp linker和evopreQ1/ mpknot以提高稳定性		1-3-bp subs； 3-bp ins			2.6~60.5 (Pr, Re)		水稻		[44]
PPE3-mpknot	nCas9(H840A)密码子优化		M-MLV密码子优化(D200N/ L603W/T330P/T306K/W31S)				1-3-bp subs； 3-bp ins			0~6.3 (Pr, Re)		水稻		[44]
pPEmax	nCas9(R221K/N394K/H840A) 密码子优化		M-MLV密码子优化，N端添加 NLS-embedded linker，C端添加异质串联NLS		epegRNA：在pegRNA 3'端添加evopreQ1		1-bp ins； 1-2-bp subs			14.58~66.67 (Re)		水稻		[45]
pPEmax- MLHdn			在pPEmax的基础上，C端添加MLH1dn，以抑制MMR途径		epegRNA：在pegRNA 3'端添加evopreQ1					0.18~2.3 (Re)
enpPE2			同pPEmax		在U6复合启动子驱动下，构成tRNA(Gly-tRNA):: pegRNA::evopreQ1:: HDV表达盒					64.58~77(Re)
pINPE2	nCas9(H840A)密码子优化， N端添加小肽		M-MLV C端添加异质串联NLSs		epegRNA：在pegRNA 3'端添加evopreQ1					0.01~0.8 (Re)
phyPE2	nCas9(H840A)密码子优化		M-MLV N端添加DBD linker (hRad51-ssDBD)		epegRNA：在pegRNA 3'端添加evopreQ1					<0.1(Re)
ePE3max	nCas9(R221K/N394K/H840A) 密码子优化		M-MLV密码子优化(D200N/ L603W/T330P/T306K/W31S)		U6复合启动子驱动的表达盒中添加tRNA(Gly-tRNA)和HDV核酶，并添加nicking sgRNA		1-bp ins； 1-3-bp subs			~40(Re)		水稻		[46]
ePE5max	nCas9(R221K/N394K/H840A) 密码子优化		M-MLV C端添加MLH1dn，以抑制MMR途径		U6复合启动子驱动的表达盒中添加tRNA(Gly-tRNA)和HDV核酶，并添加nicking sgRNA		1-bp ins； 1-3-bp subs			~40(纯合率提高)(Re)		水稻		[46]
PrimeRoot	nCas9(H840A)密码子优化		M-MLV去除RNase H结构域，并添加重组酶		采用“双ePPE”策略，使用了两个相邻的epegRNA，每个模板都包含一个仅与另一个epegRNA模板具有同源性的RT模板		720-bp，1.4-kb，4.9-kb, 7.7-kb，11.1-kb ins			0~8.3 (Pr, Re)		水稻		[47]
ePPE*	nCas9(H840A)密码子优化		M-MLV密码子优化(D200N/ V223A/T306K/W313F/T330P/ L603W)，去除RNase H结构域并添加病毒核衣壳(NC)蛋白；优化核定位信号		epegRNA：在pegRNA 3’端添加evopreQ1		1-3-bp subs； 3-6-bp del； 4-bp ins			0~18.9 (Pr, Re)		小麦		[48]
ePPEmax	nCas9(H840A)密码子优化
ePPEmax*	nCas9(R221K/N394K/H840A) 密码子优化
ePPEplus	nCas9(R221K/N394K/H840A) 密码子优化
CMPE- ePPEplus	Csy4核糖核酸内切酶				串联多个(e)pegRNA		6- bp del; 1-bp subs			2~21.6; 19.6~86.3 (Pr, Re)
ePE5max (玉米)	nCas9(R221K/N394K/H840A) 密码子优化		M-MLV C端添加MLH1dn玉米同源物，以抑制MMR途径		U6复合启动子驱动的双pegRNA表达盒中添加tRNA(Gly-tRNA)和HDV核酶		1-bp subs			Homo: 1.4~3 hetero: 15~75 (Re)		玉米		[49]
pCXPE01, pCXPE02, pCXPE03	nCas9(H840A)密码子优化		M-MLV植物密码子优化		原始的pegRNA		2-bp subs； 1-3-bp del			0.025~1.66; 2.6 (Pr)		番茄		[50]
pPPED	nCas9(H840A)密码子优化		M-MLV植物密码子优linker序列类型优化		原始的pegRNA		2-bp subs			0.06 (Pr)		本氏烟草		[51]
系统名称		系统组件						编辑类型	编辑效率 (%)		物种		参考文献
系统名称		Cas9 Nickcase		逆转录酶		pegRNA		编辑类型	编辑效率 (%)		物种
pPPEDs								2-bp subs； 25-66-bp ins	0.07±0.12 (Re)		拟南芥
pPPEM								2-bp subs；25-66-bp ins	0.7~2.2 (Pr)		水稻
pAct-PPE		nCas9(H840A)密码子优化		M-MLV植物密码子优化		原始的pegRNA		3-bp subs	0~5 (Re)		小立碗藓、马铃薯		[52]
PPE2 PPE3 PPE3b		nCas9(H840A)密码子优化		M-MLV植物密码子优化		原始的pegRNA和nicking sgRNA		1-bp subs	0.2~0.5 (Pr)		花生，鹰嘴豆，豇豆		[53]
PE-Nt2		nCas9(H840A)密码子优化		M-MLV通过linker在nCas9 C 端融合		pegRNA与nesgRNA (用于切割非编辑链的增强sgRNA)通过tRNA序列连接		1-bp subs	0~1.4 (Re)		烟草		[54]
PE-Nt3				M-MLV通过linker在nCas9 N 端融合					1.1~7.5 (Re)
PE-Nt4				M-MLV通过linker在nCas9 N 端融合					1.3~16.3 (Re)
PE2 (v2)		nCas9(H840A)密码子优化		在nCas9-M-MLV 融合蛋白N端添加T5外切酶		原始的pegRNA、双pegRNA优化、 PBS的长度优化		4-bp ins； 4-bp del； 1−2-bp subs	3.5~48.65 (Pr, Re)		水稻		[55]
PE3-HS/AS/ DS		nCas9(H840A)密码子优化		M-MLV植物密码子优化；潮霉素代理系统、除草剂代理系统和潮霉素+除草剂双代理系统		tRNA-pegRNA(n)- tRNA-sgRNA(n)- tRNA		1-7-bp subs	1.3~54.2 (Re)		水稻		[56]
PE-DSM vector		nCas9(H840A)密码子优化		潮霉素+除草剂双代理多基因编辑系统		tRNA-pegRNA(n)- tRNA-sgRNA(n)- tRNA		1-7-bp subs	1.8~45.8 (Re)		水稻		[56]
DPE, TPE, QPE		nCas9(R221K/N394K/H840A)密码子优化		同ePE3max		pegRNA(n)-ngRNA(n)通过Gateway连接		2-3-bp subs, 28-bp ins	57.14 (Re)		水稻		[57]
-		nCas9(R221K/N394K/H840A) 密码子优化		同ePE5max		epegRNA：在pegRNA 3’端添加evopreQ1		4-bp ins	9.7 (Re)		番茄、拟南芥		[58]
GRAND		nCas9(H840A)密码子优化		M-MLV植物密码子优化		RTT部分对齐，但与双pegRNA中的目标序列非同源		46-bp subs	0.59~9.88 (Pr, Re)		水稻		[59]
PE5max		nCas9(R221K/N394K/H840A) 密码子优化		M-MLV C端添加MLH1dn，以抑制MMR途径		U6复合启动子驱动的双pegRNA表达盒中添加tRNA(Gly-tRNA)和HDV核酶		30-bp ins	47 (Re)		水稻		[60]

Cas9变体	Cas蛋白大小（kb）	预测的PE载体大小（kb）	参考文献
SpCas9	4.2	6.2	[107]
SaCas9	3.2	5.2	[9]
CjCas9	3.0	5.0	[108]
Casϕ	2.1~2.4	4.1~4.4	[62]
Cas12f	1.2~1.8	3.2~3.8	[63]
CasX	<3.0	<5.0	[19]
Cas13j	1.2~1.5	3.2~3.5	[109]