Research progress and biotechnological applications of the prime editing

doi:10.12211/2096-8280.2023-038

Abstract

Abstract:

Prime Editor (PE) is an innovative gene editing tool based on the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) system, which has revolutionized multiple fields, including genetics, medicine, and agriculture. Emerging as a successor to Base Editor (BE), PE has gained worldwide attention due to its ability to introduce base substitutions, insertions, and deletions without causing double-strand DNA breaks, which significantly reduces the risk of off-target effect and unwanted genetic change. Notwithstanding its immense potential, researchers need to address PE's long encoding sequence and low editing efficiency for its maximal applications. Researchers have been working relentlessly to explore and enhance the editing efficiency and safety of PE by modifying its protein scaffold, optimizing the guide RNA design, and identifying cellular factors that influence its activity. Improved PE variants have been developed with enhanced accuracy and efficiency as well as decreased off-target effect when compared with their initial versions, demonstrating their potential in gene editing-related applications. Several strategies have been investigated to enhance PE performance, including: ① Modifying the structure of PE proteins to increase their efficiency, specificity, and binding affinity, thereby significantly improving their editing activity. ② Optimizing the design of pegRNAs, such as modifying the length, composition, or structure, that can boost PE's editing efficiency. ③ Identifying and manipulating cellular factors, such as proteins and RNAs, that bear functional relationships with the PE system, thus greatly enhancing its gene editing capabilities. ④ Developing automated design tools to facilitate the customization of the PE system for specific applications, vastly improving its practicality in research and clinical settings. Finally, this article summarizes the applications of PE in engineering animals and plants and developing gene therapy. Despite much room for further improvement in PE, significant advances have been made in improving its editing efficiency and safety. The rapid development of Cas9 and BE for treating genetic diseases stands as compelling testimony to the potential of PE in advancing gene editing technologies and applications. With continued research and development, PE holds great promise for improving human health and well-being.

Key words: prime editing, gene editing, gene therapy, CRIPSR/Cas

摘要：

引导编辑器（prime editor，PE）是继碱基编辑器（base editor，BE）之后新问世的基于CRISPR/Cas（clustered regularly interspaced short palindromic repeats/CRISPR-associated）系统的基因编辑工具，可以在不造成DNA双链断裂的情况下引入碱基替换、插入和删除。PE因其全面的编辑能力，问世即受到全球学者的广泛关注，然而PE表达盒编码较长（>6 kb）、编辑效率较低等问题也亟待研究人员解决。PE的研究方向与BE有许多相似之处，本文首先梳理了学界对PE本身编辑效率和安全性的探索；然后重点介绍了PE效应蛋白、pegRNA和其他细胞因子三个方面对PE的改进手段，以及为方便PE应用而开发的自动化设计工具；最后梳理了PE在动植物以及基因治疗中的应用。方兴未艾的PE领域尽管还难称完善，但在提高编辑效率和改进安全性等方面已取得了许多重要进展。鉴于Cas9、BE等基因编辑工具已广泛应用于遗传病疗法，PE走向遗传病治疗值得期待。

关键词: 引导编辑, 基因编辑, 基因治疗, CRISPR/Cas

CLC Number:

Q812

Zhimeng XU, Zhen XIE. Research progress and biotechnological applications of the prime editing[J]. Synthetic Biology Journal, 2024, 5(1): 1-15.

许志锰, 谢震. 引导编辑研究进展及其应用[J]. 合成生物学, 2024, 5(1): 1-15.

Figures/Tables 7

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工具名称	特点	实验细胞系	链接	参考文献
DeepPE	给出DeepPE、PE_type、PE_position三个模型的结果	HEK293FT、HCT116、MDA-MB-231	http://deepcrispr.info/DeepPE	[12]
DeepPrime	将DeepPE扩展到7种细胞系、8种PE	HEK293FT、HCT116、MDA-MB-231、HeLa、DLD1、A549、NIH3T3	http://deepcrispr.info/DeepPrime/	[21]
PRIDICT	包括ClinVar突变体数据，但输入格式较为复杂	HEK293T、U2OS、K562	https://www.pridict.it/	[22]
MinsePIE	Spacer、PBS等长度可手动调整，自由度较高	HEK293T、HAP1	https://elixir.ut.ee/minsepie/	[23]

工具名称	特点	实验细胞系	链接	参考文献
DeepPE	给出DeepPE、PE_type、PE_position三个模型的结果	HEK293FT、HCT116、MDA-MB-231	http://deepcrispr.info/DeepPE	[12]
DeepPrime	将DeepPE扩展到7种细胞系、8种PE	HEK293FT、HCT116、MDA-MB-231、HeLa、DLD1、A549、NIH3T3	http://deepcrispr.info/DeepPrime/	[21]
PRIDICT	包括ClinVar突变体数据，但输入格式较为复杂	HEK293T、U2OS、K562	https://www.pridict.it/	[22]
MinsePIE	Spacer、PBS等长度可手动调整，自由度较高	HEK293T、HAP1	https://elixir.ut.ee/minsepie/	[23]

名称	优化点	优化结果	实验细胞系或动植物模型	参考文献
PE2*	优化NLS	提高编辑效率	HEK293T	[30]
PEmax	更换为SpCas9（R221K/N394K/H840A），优化NLS，优化RT密码子	提高编辑效率	HEK293T、HeLa	[31]
hyPE2	融合Rad51 DNA结合结构域	提高编辑效率	HEK293T、HCT116	[32]
CMP-PE-V1	融合染色体操纵肽	提高编辑效率	NIH/3T3、C2C12细胞系，C57BL/6N、ICR小鼠	[33]
IN-PE2	融合短肽	提高编辑效率	HEK293T、U2OS、HCT116	[34]
PASTE	融合Bxb1丝氨酸整合酶	可在非分裂细胞基因组中整合超大片段	HEK293FT、HepG2、K562、原代人外周血CD8 T细胞、原代人肝细胞	[35]
ePE	引入Csy4蛋白	提高编辑效率	HEK293T、HeLa、N2a	[36]
WT-PE、PEn	Cas9n更换为野生型Cas9	增强大片段删除或易位编辑效率，证明了NHEJ途径可用于PE	HEK293T、HeLa、HCT116	[37-38]
未命名	对Cas9n引入N863A或N854A突变	减少indel	HEK293T、HeLa、K562	[39]
GENEWRITE	更换LINE-1 RT	实现大片段插入	E.coli	[40]
ePPE	融合病毒核衣壳蛋白，删除RNaseH结构域	提高编辑效率	Zhonghua11、Kenong199	[41]
PE-P3	颠倒Cas9n和M-MLV RT	提高编辑效率	Nipponbare	[42]
PE2△Rnh	删除RNaseH结构域	缩小PE尺寸	HEK293T、Hepa1-6、RAW264.7细胞系，C57BL/6J、FVB/NJ小鼠	[41,43-45]
sPE	拆分Cas9n和M-MLV RT	方便递送和优化	HEK293T	[46]
PE2-VQR、PE2-VRQR、PE2-VRER、PE2-NG、 PE2-SpG、PE2-SpRY	更换其他Cas9变体	松弛PAM要求，扩展可编辑范围	HEK293T	[47]
SaPE2	SpCas9更换为SaCas9	更改PAM要求	HEK293T、HeLa、K562	[30-31,44]
CjCas9-PE	SpCas9更换为CjCas9	更改PAM要求	HEK293T	[44]
FnCas9-PE	SpCas9更换为FnCas9	更改PAM要求	HEK293T、HeLa、K562、U2OS	[48]
SauriCas9-PE	SpCas9更换为SauriCas9	更改PAM要求	HEK293T	[44]

名称	优化点	优化结果	实验细胞系或动植物模型	参考文献
PE2*	优化NLS	提高编辑效率	HEK293T	[30]
PEmax	更换为SpCas9（R221K/N394K/H840A），优化NLS，优化RT密码子	提高编辑效率	HEK293T、HeLa	[31]
hyPE2	融合Rad51 DNA结合结构域	提高编辑效率	HEK293T、HCT116	[32]
CMP-PE-V1	融合染色体操纵肽	提高编辑效率	NIH/3T3、C2C12细胞系，C57BL/6N、ICR小鼠	[33]
IN-PE2	融合短肽	提高编辑效率	HEK293T、U2OS、HCT116	[34]
PASTE	融合Bxb1丝氨酸整合酶	可在非分裂细胞基因组中整合超大片段	HEK293FT、HepG2、K562、原代人外周血CD8 T细胞、原代人肝细胞	[35]
ePE	引入Csy4蛋白	提高编辑效率	HEK293T、HeLa、N2a	[36]
WT-PE、PEn	Cas9n更换为野生型Cas9	增强大片段删除或易位编辑效率，证明了NHEJ途径可用于PE	HEK293T、HeLa、HCT116	[37-38]
未命名	对Cas9n引入N863A或N854A突变	减少indel	HEK293T、HeLa、K562	[39]
GENEWRITE	更换LINE-1 RT	实现大片段插入	E.coli	[40]
ePPE	融合病毒核衣壳蛋白，删除RNaseH结构域	提高编辑效率	Zhonghua11、Kenong199	[41]
PE-P3	颠倒Cas9n和M-MLV RT	提高编辑效率	Nipponbare	[42]
PE2△Rnh	删除RNaseH结构域	缩小PE尺寸	HEK293T、Hepa1-6、RAW264.7细胞系，C57BL/6J、FVB/NJ小鼠	[41,43-45]
sPE	拆分Cas9n和M-MLV RT	方便递送和优化	HEK293T	[46]
PE2-VQR、PE2-VRQR、PE2-VRER、PE2-NG、 PE2-SpG、PE2-SpRY	更换其他Cas9变体	松弛PAM要求，扩展可编辑范围	HEK293T	[47]
SaPE2	SpCas9更换为SaCas9	更改PAM要求	HEK293T、HeLa、K562	[30-31,44]
CjCas9-PE	SpCas9更换为CjCas9	更改PAM要求	HEK293T	[44]
FnCas9-PE	SpCas9更换为FnCas9	更改PAM要求	HEK293T、HeLa、K562、U2OS	[48]
SauriCas9-PE	SpCas9更换为SauriCas9	更改PAM要求	HEK293T	[44]

名称	pegRNA优化方式	参考文献
epegRNA	3′端添加tevopreQ₁或mpknot基序以防降解	[53]
xrPE	3′端添加xrRNA继续以防降解	[54]
G-PE	3′端添加G-quadruplex基序以防降解	[55]
ePE	3′端添加Csy4基序以防环化	[36]
apegRNA	优化hairpin	[56]
spegRNA	RT模板引入统一突变以改善二级结构	[56]