基因编辑中定点敲入技术的研究进展

doi:10.16288/j.yczz.25-076

遗传 ›› 2026, Vol. 48 ›› Issue (2): 128-141.doi: 10.16288/j.yczz.25-076

基因编辑中定点敲入技术的研究进展

王梓豪(), 杨昭庆()

中国医学科学院&北京协和医学院医学生物学研究所医学遗传室，昆明 650118

收稿日期:2025-04-22 修回日期:2025-06-20 出版日期:2026-07-01 发布日期:2025-07-01
通讯作者: 杨昭庆，博士，研究员，研究方向：遗传学。E-mail: zyang@imbcams.com.cn
作者简介:王梓豪，硕士，助理研究员，研究方向：遗传学。E-mail: zihaowang_bio@163.com
基金资助:
协和创新工程(2021-I2M-1-024);云南省院士及科技领军人才专项-创新疫苗载体及创新疫苗研发(202405AB350002);云南省高层次卫生健康技术人才培养专项(L-2018003)

Advances in site-specific knock-in techniques for gene editing

Zihao Wang(), Zhaoqing Yang()

Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China

Received:2025-04-22 Revised:2025-06-20 Published:2026-07-01 Online:2025-07-01
Supported by:
CAMS Innovation Fund for Medical Sciences (CIFMS)(2021-I2M-1-024);Innovative Vaccine Vectors and Innovative Vaccine Development(202405AB350002);Yunnan Provincial Special Project for Cultivation of High-Level Health Technology Talents(L-2018003)

摘要/Abstract

摘要：

基因定点敲入技术是基因工程和基因治疗的核心工具，旨在避免传统随机整合方法的不可控性及异质性等问题，但是在实际的应用过程中却长期受编辑过程中的脱靶活性和低效性的限制。近年来，位点特异性重组酶系统(如Bxb1整合酶)和可编程核酸酶系统(如CRISPR/Cas9)的发展显著提升了基因敲入的精确性与效率。特别是Cas9-Bxb1整合酶系统的出现，实现了5~43 kb大片段DNA在基因组安全港(genomic safe harbor，GSH)位点的靶向整合，为疾病模型构建、基因功能研究和临床治疗提供了突破性平台。本文系统综述了位点特异性重组酶与核酸酶系统的研究进展，探讨了GSH的筛选策略及多组学数据在优化预测模型中的应用，并对比分析了twinPE+Bxb1与PASTE系统的优势与局限性。未来研究需聚焦于开发低脱靶活性的新型整合酶、优化无双链断裂(double strand break，DSB)编辑技术，以及建立跨物种GSH数据库，以进一步推动基因编辑技术在精准医学和合成生物学中的应用。

关键词: 基因定点敲入, 基因组安全港, CRISPR/Cas9, 先导编辑, Bxb1整合酶

Abstract:

Gene-targeted knock-in technology serves as a cornerstone tool in genetic engineering and gene therapy, designed to circumvent the unpredictability and heterogeneityassociated with conventional random integration methods. However, its practical application has long been constrained by off-target activity and low efficiency during the editing process. Recent advances in site-specific recombinase systems (e.g., Bxb1 integrase) and programmable nuclease systems (e.g., CRISPR/Cas9) have significantly enhanced the precision and efficiency of gene knock-in. Notably, the Cas9-Bxb1 integrase system enables targeted integration of large DNA fragments (5−43 kb) into genomic safe harbor (GSH) sites, offering a transformative platform for disease modeling, functional genomics, and clinical therapeutics. This review systematically summarizes the progress of site-specific recombinase and nuclease systems, discusses GSH screening strategies and the role of multi-omics data in optimizing predictive models, and compares the strengths and limitations of twinPE+Bxb1 and PASTE systems. Future research should focus on developing novel integrases with low off-target activity, refining DSB-free editing technologies, and establishing cross-species GSH databases to advance applications in precision medicine and synthetic biology.

Key words: gene targeted knock-in, genomic safe harbor, CRISPR/Cas9, prime editing, Bxb1 integrase

王梓豪, 杨昭庆. 基因编辑中定点敲入技术的研究进展[J]. 遗传, 2026, 48(2): 128-141.

Zihao Wang, Zhaoqing Yang. Advances in site-specific knock-in techniques for gene editing[J]. Hereditas(Beijing), 2026, 48(2): 128-141.

图/表 2

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类别	常用工具	机制	优点	缺点	应用场景	随机/定点
转座酶	piggyBac、 sleeping beauty	通过“剪切-粘贴”机制敲入DNA	1. 可敲入大片段DNA； 2. 可在多种细胞类型中高效整合到基因组	1. 敲入位点的随机性可能导致基因突变； 2. 可能导致敲入多个拷贝，增加基因组的不稳定性	适合大规模基因敲入	随机
逆转录病毒	γ-逆转录病毒	以RNA为模板，通过逆转录酶合成DNA后整合到宿主基因组	1. 高效感染分裂细胞； 2. 携带较大片段DNA； 3. 稳定整合到宿主基因组	1. 敲入位点的随机性可能导致基因突变； 2. 安全性问题，具有遗传毒性和免疫原性； 3.倾向于整合附近的转录单位	适合分裂细胞的基因传递
慢病毒载体	基于HIV的病毒载体	以RNA为模板，通过逆转录酶合成DNA后整合到宿主基因组	1. 可感染分裂和非分裂细胞； 2. 携带较大片段DNA； 3. 长期稳定表达	1. 敲入位点的随机性可能导致基因突变； 2. 安全性问题，具有遗传毒性和免疫原性； 3.倾向于整合附近的转录单位	适合分裂和非分裂细胞，应用广泛
重组酶	Cre-lox、Flp-FRT、Bxb1	通过识别特定DNA序列并催化DNA断裂和链交换及重接	高度特异性，可精确敲入目标位点	需要特定的识别序列，要预先敲入识别位点	适合精确敲入，但需要预先设计	半随机
核酸酶	ZFN、TALEN和CRISPR/Cas9	通过切割DNA形成DSB，诱导修复机制	高特异性切割DNA，可用于精确基因编辑	1. 可能产生非特异性DNA断裂，导致细胞死亡或基因组不稳定； 2. 引导RNA需要精确地设计和优化以避免脱靶效应	适合精确基因编辑	定点
核酸酶	PE	单链切口修复，不形成DSB	1. 可实现单碱基替换、小片段敲入和删除； 2. 不依赖DSB，减少基因组不稳定性	1. 仅能实现小片段的敲入和缺失，不容易进行多核苷酸的操作； 2. 可能产生脱靶效应	适合精确基因编辑	定点

基因编辑中定点敲入技术的研究进展

Advances in site-specific knock-in techniques for gene editing

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