转座元件系统介导的大片段DNA定向插入技术研究进展

doi:10.3864/j.issn.0578-1752.2026.06.001

Abstract

Abstract:

The rapidly evolving genome editing technologies have demonstrated strong application potential in animal and plant breeding, microbial engineering, and basic scientific research. Current genome editing techniques allow for the insertion, deletion, and substitution of single or multiple nucleotides at specific genomic targets across a wide range of species. However, editing types involving large DNA fragment insertion or replacement still face technical bottlenecks, such as low efficiency and fidelity, as well as difficulties in donor delivery. These limitations restrict the application of gene editing in important scenarios, including multigene stacking with genetic linkage, precise replacement of favorable alleles, and targeted integration of DNA fragments at genomic safe harbors. Transposable elements, as mobile genetic elements widely present in biological genomes, offer a novel approach to overcoming these challenges due to their inherent mobility and large DNA cargo capacity. They hold promise for being engineered into key molecular tools for precise large DNA fragment editing. This review summarizes recent advances in targeted large DNA fragment insertion technologies based on transposable elements, focusing on the application status and prospects of prokaryotic-derived CRISPR-associated transposons (CAST) and certain DNA transposons and retrotransposons in eukaryotes. Prokaryotic-derived CAST systems have shown outstanding performance, enabling efficient large fragment integration in prokaryotes and, after optimization, also achieving large fragment insertion in eukaryotic cells. In eukaryotes, engineered DNA transposons such as mPing/Pong and retrotransposon-related tools like R2 and L1 have been utilized for large DNA fragment insertion in animals and plants. At the same time, the field of transposon-based large DNA fragment insertion faces challenges. On the one hand, the cross-species adaptability of transposable elements is limited, making it difficult for some elements to function when transferred to other species. On the other hand, the large size or multiplicity of protein components involved leads to low delivery efficiency in certain types of eukaryotic cells. Additionally, some systems carry safety risks, such as stimulating the mammalian immune system and triggering inflammatory responses. Future research may focus on the discovery of novel transposable elements, engineering of transposases, development of new delivery vectors, and in-depth elucidation of transposition mechanisms, in order to provide key technical support for establishing efficient and safe large fragment editing technologies. This will contribute to foundational innovations in crop genetic improvement, gene therapy, and microbial genome editing.

Key words: genome editing, DNA transposon, R2 retrotransposon, CRISPR-associated transposon, targeted DNA insertion, crop genetic improvement

ZHAO ZiJie, SONG Hao, DONG XiaoOu, WAN JianMin. Progress in Transposable Element-Assisted Targeted Insertion of Large DNA Fragments[J].Scientia Agricultura Sinica, 2026, 59(6): 1141-1156.

Figures/Tables 6

Fig. 1

Fig. 2

Table 1

Reported transposable element-assisted targeted insertion of large DNA fragments"

转座元件名称 Transposable elements	敲入工具名称 Knock- in tools	操作物种 Experimental species	敲入片段最大长度 Maximum length of knock-in fragment (kb)	敲入效率 Knock-in efficiency (%)	敲入原理 Knock-in principle	参考文献 References
L1	CREATE	Huh7细胞 Huh7 cells	1.1	1.5	设计一对sgRNA引导nCas9切割靶点上下游DNA，释放的末端与L1 mRNA的PBS互补配对，介导搭载外源基因的L1启动TPRT机制，实现片段定点插入 Design dual sgRNAs to guide nCas9 to cleave the DNA upstream and downstream of the target site. The released ends form complementary pairing with the primer binding site (PBS) of L1 mRNA, mediating the L1 carrying the exogenous gene to initiate the target-primed reverse transcription (TPRT) mechanism, thereby achieving targeted insertion of the fragment	[73]
mPing/Pong	TATSI	拟南芥 Arabidopsis	8.6	8.3	CRISPR/Cas系统产生DSB后，Pong转座酶的ORF1/ORF2蛋白将mPing转座子插入靶DNA位点 After the CRISPR/Cas system generates double-strand breaks (DSB), the ORF1/ORF2 proteins of Pong transposase insert the mPing transposon into the target DNA site	[51]
R2Tg	PRINT	ARPE-19细胞 ARPE-19 cells	4.5	/	R2Tg蛋白通过TPRT机制实现RNA模板在28S rDNA安全港位点实现大片段整合 The R2Tg protein realizes large-fragment integration of the RNA template at the 28S rDNA safe-harbor locus through the TPRT mechanism	[58]
R2Tg	en-R2Tg	HEK293T细胞 HEK293T cells	1.5	1.0	优化R2Tg蛋白结构与供体RNA模板，实现全RNA递送的安全港位点的大片段定向插入 Optimize the structure of R2Tg protein and the donor RNA template to achieve targeted insertion of large fragments at the safe-harbor locus via all-RNA delivery	[59]
R2Tg	/	HEK293FT细胞 HEK293FT cells	3.0	1.0	通过精简供体UTR序列，大幅减少了供体片段的插入痕迹，并增加化学修饰，实现全RNA递送的安全港位点敲入 By streamlining the donor UTR sequence, the insertion scars of the donor fragment are significantly reduced, and chemical modifications are added to achieve knock-in at the safe-harbor locus via all-RNA delivery	[60]
R2Tocc	STITCHR	哺乳动物细胞 Mammalian cells	12.7	9.0	通过重新设计供体模板的同源臂序列位置，将nCas9与R2Tocc捆绑，实现非28S rDNA位点的无痕插入 By redesigning the position of the homologous arm sequences of the donor template and fusing nCas9 with R2Tocc, scarless insertion at non-28S rDNA loci is achieved	[62]
Tn7-like	VchCAST	大肠杆菌 E. coli	10.1	1.0	仅有靶向能力的CRISPR/Cascade引导Tn7样转座子在靶点下游处进行供体的靶向整合 The CRISPR/Cascade with only targeting capability guides the Tn7-like transposon to perform targeted integration of the donor downstream of the target site	[35]
Tn7-like	ShCAST	大肠杆菌 E. coli	≈10.0	45.0	仅有靶向能力的CRISPR/Cas12k引导Tn7样转座子在靶点下游处进行供体的靶向整合 The CRISPR/Cas12k with only targeting capability guides the Tn7-like transposon to perform targeted integration of the donor downstream of the target site	[34]
Tn7-like	INTEGRATE	大肠杆菌 E. coli	10.0	99.0	建立单质粒I-F型CAST系统，实现10 kb片段近100%效率的无标记和高精度定向整合 Establish an all-in-one plasmid Type I-F CAST system to achieve marker-free, high-precision targeted integration of 10 kb fragments with nearly 100% efficiency	[39]
Tn7-like	ShHELIX	大肠杆菌 E. coli	9.8	70.0	融合归巢核酸内切缺刻酶（nHE）与TnsB，提升V-K型CAST的插入效率与精度，并减少质粒骨架共整合的频率 Fuse homing endonuclease nickase (nHE) with TnsB to improve the insertion efficiency and precision of Type V-K CAST, and reduce the frequency of plasmid backbone co-integration	[40]
Tn7-like	evoCAST	HEK293T细胞 HEK293T cells	14.8	8.0	通过连续的噬菌体辅助进化手段，将进化后的TnsB与其余优化的CAST组分结合，实现真核细胞中的大片段定点单向整合 Through continuous phage-assisted continuous evolution (PACE), combine the evolved TnsB with other optimized CAST components to achieve targeted unidirectional integration of large fragments in eukaryotic cells	[42]
Tn7-like	MetaEdit	拟杆菌 B. thetaiotaomicron	7.5	/	通过接合型大肠杆菌将可移动CAST元件递送至小鼠肠道菌群 Deliver mobilizable CAST elements to the mouse gut microbiota via conjugative E. coli	[43]
PiggyBac	FiCAT	HEK293T细胞 HEK293T cells	8.0	≈0.3	将PiggyBac与Cas9蛋白融合，通过靶向DSB实现片段整合 Fuse PiggyBac with Cas9 protein to achieve fragment integration through targeted DSB	[52]

Table 1

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Progress in Transposable Element-Assisted Targeted Insertion of Large DNA Fragments

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