Evolution, Applications, and Perspectives of Technologies for Genetically Engineered Mouse Generation

doi:10.12300/j.issn.1674-5817.2026.028

Abstract

Abstract:

Genetically engineered mice are important tools for elucidating gene function and disease mechanisms and for advancing drug development. With the growing demand in biomedical research for complex genetic modifications, rapid model generation, and high-throughput functional analysis, technologies for generating genetically engineered mice continue to evolve. This review systematically summarizes the evolution and applications of these technologies, with a focus on conventional approaches, including early transgenic technologies and embryonic stem cell (ESC)-based homologous recombination for gene targeting, as well as the use of random mutagenesis approaches, such as N-ethyl-N-nitrosourea (ENU) mutagenesis and transposon insertion, in forward genetic screening. It further discusses the principles and characteristics of programmable nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas systems, as well as their applications in generating genetically engineered mice. With advances in genome editing, Cas9-derived systems, base editing, prime editing, and RNA editing further improve editing precision, expand the repertoire of genetic modifications and precise regulatory approaches, and provide additional options for generating genetically engineered mice. This review also compares the advantages, limitations, and suitability of different technologies for specific applications and summarizes quality-control strategies addressing practical issues that may affect model reliability, including off-target effects, mosaicism, genetic background, and genetic stability. Finally, future directions in technologies for generating genetically engineered mice are discussed to inform the generation and application of related models.

Key words: Genetically engineered mice, Gene targeting technology, Random mutagenesis models, CRISPR/Cas system

CLC Number:

R-332
Q95-33

WU Jianfeng,SHAO Zhiqiang,HE Ying,et al. Evolution, Applications, and Perspectives of Technologies for Genetically Engineered Mouse Generation[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 759-770. DOI: 10.12300/j.issn.1674-5817.2026.028.

Figures/Tables 2

References 65

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技术类型 Technology	编辑原理 Editing principle	是否依赖DSB Depen-dence on DSB	可实现的突变类型 Types of mutations achievable	编辑效率 Editing efficiency	构建周期 Model genera-tion time	构建成本 Cost	品系适应性 Strain compat-ibility	多基因编辑能力 Multiplex editing capability	脱靶风险 Off-target risk	主要优势 Key advantage	主要局限 Key limitation	典型应用 Typical application
胚胎干细胞同源重组 ESC-based homologous recombination	同源重组介导靶位点定向替换	否	KO、KI、点突变等均可实现	低	长（6～12个月）	高	受限（前期主要为129等品系）	低	低	精确性高，遗传结果稳定	周期长、成本高	精确遗传修饰、经典KO/KI模型
ENU/转座子诱变 ENU/Transposon mutagenesis	ENU诱导碱基烷基化引起点突变；转座子在基因组中随机或偏好性插入导致基因功能破坏	否	点突变（ENU）、插入突变（转座子）	高（突变率高）	长	中	高	高（但随机性强）	不适用于传统脱靶概念，且存在随机突变背景	无须预设靶点，可进行正向遗传筛选	位点鉴定困难、表型归因复杂	正向遗传筛选、新基因发现
锌指核酸酶 Zinc finger nucleases	蛋白识别DNA序列并由FokⅠ诱导产生DSB	是	KO、KI、点突变等均可实现	中	中	高	高	低	中	开创性定点编辑工具	设计复杂、成本高、专利限制	特定靶点编辑或已有体系延续
转录激活因子样效应物核酸酶 Transcription activator-like effector nuclease	转录激活因子样效应物识别DNA序列并由FokⅠ诱导产生DSB	是	KO、KI、点突变等均可实现	中～高	中	中～高	高	低	低	特异性较高、识别规则明确	构建复杂、递送受限	高特异性需求或复杂靶点编辑
CRISPR/Cas系统 CRISPR/Cas system	sgRNA引导Cas9在靶位点产生DSB	是	KO、KI、点突变等均可实现	高	短（2～3个月）	低	高	高（多条sgRNA实现多靶向）	中	灵活、高效、适用于多基因编辑	存在脱靶及嵌合性风险	常规基因工程小鼠构建首选
碱基编辑 Base editing	脱氨酶介导碱基转换	否	主要为点突变	高	短	中	高	中	低～中	无DSB、高精度	编辑窗口受限	单碱基突变疾病模型
引导编辑 Prime editing	逆转录酶介导的靶位点序列改写	否	片段序列精准插入/缺失/替换	中	短～中	中	高	中	低	突变类型多样、灵活性高	编辑效率相对较低	复杂突变或非编码区精细编辑模型

技术类型 Technology	编辑原理 Editing principle	是否依赖DSB Depen-dence on DSB	可实现的突变类型 Types of mutations achievable	编辑效率 Editing efficiency	构建周期 Model genera-tion time	构建成本 Cost	品系适应性 Strain compat-ibility	多基因编辑能力 Multiplex editing capability	脱靶风险 Off-target risk	主要优势 Key advantage	主要局限 Key limitation	典型应用 Typical application
胚胎干细胞同源重组 ESC-based homologous recombination	同源重组介导靶位点定向替换	否	KO、KI、点突变等均可实现	低	长（6～12个月）	高	受限（前期主要为129等品系）	低	低	精确性高，遗传结果稳定	周期长、成本高	精确遗传修饰、经典KO/KI模型
ENU/转座子诱变 ENU/Transposon mutagenesis	ENU诱导碱基烷基化引起点突变；转座子在基因组中随机或偏好性插入导致基因功能破坏	否	点突变（ENU）、插入突变（转座子）	高（突变率高）	长	中	高	高（但随机性强）	不适用于传统脱靶概念，且存在随机突变背景	无须预设靶点，可进行正向遗传筛选	位点鉴定困难、表型归因复杂	正向遗传筛选、新基因发现
锌指核酸酶 Zinc finger nucleases	蛋白识别DNA序列并由FokⅠ诱导产生DSB	是	KO、KI、点突变等均可实现	中	中	高	高	低	中	开创性定点编辑工具	设计复杂、成本高、专利限制	特定靶点编辑或已有体系延续
转录激活因子样效应物核酸酶 Transcription activator-like effector nuclease	转录激活因子样效应物识别DNA序列并由FokⅠ诱导产生DSB	是	KO、KI、点突变等均可实现	中～高	中	中～高	高	低	低	特异性较高、识别规则明确	构建复杂、递送受限	高特异性需求或复杂靶点编辑
CRISPR/Cas系统 CRISPR/Cas system	sgRNA引导Cas9在靶位点产生DSB	是	KO、KI、点突变等均可实现	高	短（2～3个月）	低	高	高（多条sgRNA实现多靶向）	中	灵活、高效、适用于多基因编辑	存在脱靶及嵌合性风险	常规基因工程小鼠构建首选
碱基编辑 Base editing	脱氨酶介导碱基转换	否	主要为点突变	高	短	中	高	中	低～中	无DSB、高精度	编辑窗口受限	单碱基突变疾病模型
引导编辑 Prime editing	逆转录酶介导的靶位点序列改写	否	片段序列精准插入/缺失/替换	中	短～中	中	高	中	低	突变类型多样、灵活性高	编辑效率相对较低	复杂突变或非编码区精细编辑模型