CRISPR-Cas9基因编辑技术及其在家禽中的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2023-1135

摘要/Abstract

摘要：

CRISPR-Cas系统是细菌或古细菌内抵御病毒再次入侵的一种适应性免疫系统，能够对靶向核酸进行切割。基于Cas9蛋白及其突变体的特性，科学家开发出多样化的基因编辑工具，能够对细胞内的基因进行敲除或敲入等基因编辑操作，实现生物的遗传变异；或对DNA或RNA进行表观修饰，调控基因的表达，已广泛地应用于生命科学的研究。禽类是农业中的重要物种，为人类提供优质的蛋白质。在禽类的基因组编辑和修饰中，CRISPR-Cas9技术仍处于研究状态，且只在鸡和鹌鹑两种家禽上取得实质性进展。本文从CRISPR-Cas9系统的组成成分、传递方法、优化策略介绍了CRISPR-Cas9基因编辑技术及其在家禽生产和疾病防控中的应用，为进一步开发适用于家禽体系的CRISPR-Cas9提供理论基础。

关键词: 家禽, CRISPR-Cas9, 育种, 诊断

Abstract:

The CRISPR-Cas system, found in bacteria or archaea, serves as an adaptive immune system to counteract viral re-invasion by effectively cleaving nucleic acids. By leveraging the characteristics of Cas9 protein and its variants, researchers have devised a range of gene editing techniques that enable gene operations like gene knock-out or knock-in within cells, thereby facilitating genetic diversity in organisms. Additionally, these tools allow the manipulation of DNA or RNA through epigenetic modification to regulate gene expression, rendering them extensively employed in life science research. Poultry, as a significant agricultural species, plays a crucial role in supplying individuals with protein of superior quality. The utilization of CRISPR-Cas9 technology for gene editing and modification in avian genome is presently confined to the research phase, with remarkable advancements observed solely in chicken and quail. This paper presents an overview of CRISPR-Cas9 gene editing technology, encompassing its components, delivery methods, and optimization strategies, while also exploring its potential applications in poultry production and prevention and control of diseases. These insights lay the groundwork for the future development of CRISPR-Cas9 technology in the context of poultry system.

Key words: poultry, CRISPR-Cas9, breeding, diagnosis

肖怡梦, 杨雯, 程依依, 罗刚. CRISPR-Cas9基因编辑技术及其在家禽中的研究进展[J]. 生物技术通报, 2024, 40(5): 38-47.

XIAO Yi-meng, YANG Wen, CHENG Yi-yi, LUO Gang. CRISPR-Cas9 Gene Editing Technology and Its Research Progress in Poultry[J]. Biotechnology Bulletin, 2024, 40(5): 38-47.

图/表 3

图1 CRISPR-Cas9/dCas9系统切割双链DNA的示意图 A: CRISPR-Cas9切割靶DNA示意图。Cas9：CRISPR相关蛋白9；RuvC和HNH：Cas9蛋白中切割核酸的结构域；PAM序列：原间隔相邻基序；sgRNA：单链向导RNA，由crRNA（CRISPR RNA）和tracrRNA（反式激活crRNA）组成。B和C：CRISPR-nCas9切割靶DNA示意图。nCas9：Cas9切口酶；HNH（H840A）:失活HNH结构域的Cas9突变体；RuvC（D10A）：失活RuvC结构域的Cas9突变体。D：CRISPR-dCas9切割靶DNA示意图。HNH（H840A）和RuvC（D10A）：失活HNH和RuvC结构域的Cas9突变体

Fig. 1 Schematic diagram of the CRISPR-Cas9/dCas9 system cutting double-stranded DNA A: Schematic diagram of CRISPR-Cas9 cutting target DNA. Cas9: CRISPR-associated protein 9; RuvC and HNH: structural domains in the Cas9 protein that cleave nucleic acids; PAM sequence: protospacer adjacent motif; sgRNA: single-guide RNA, composed of crRNA（CRISPR RNA）and tracrRNA（trans-activating crRNA）. B and C: Schematic representation of CRISPR-nCas9 cleaving target DNA. nCas9: Cas9 nickase; HNH(H840A): Cas9 mutant with inactivated HNH domain; RuvC（D10A）: Cas9 mutant with inactivated RuvC domain. D: Schematic diagram of CRISPR-dCas9 cutting target DNA. HNH（H840A）and RuvC（D10A）: Cas9 mutants with inactivated HNH and RuvC structural domains

图2 禽类中CRISPR-Cas9系统的传递方式 A：PGC细胞介导的CRISPR-Cas9基因编辑。U6：gRNA的启动子；gRNA：向导RNA；CMV：启动Cas9编码基因的转录；GFP：绿色荧光蛋白；NLS：核定位序列；PGC：原始生殖细胞。B：腺病毒介导的CRISPR-Cas9基因编辑。C：STAGE介导的CRISPR-Cas9示意图。STAGE：精子转染辅助基因编辑技术；sgRNA：单链向导RNA；Cas 9 mRNA：Cas 9编码基因的信使RNA

Fig. 2 Delivery of the CRISPR-Cas9 system in avian species A: Schematic diagram of PGC-mediated CRISPR-Cas9 gene editing. U6: gRNA promoter; gRNA: guide RNA; CMV: The promoter of Cas9-encoded genes; GFP: green fluorescent protein; NLS: nuclear localization sequences; PGC: primordial germ cell. B: Schematic diagram of adenovirus-mediated CRISPR-Cas9 gene editing. C: Schematic representation of STAGE-mediated CRISPR-Cas9. STAGE: sperm transfection-assisted gene editing; sgRNA: single guide RNA; Cas 9 mRNA: messenger RNA of Cas 9-encoding genes

图3 基于CRISPR-Cas9系统的NASBACC的诊断原理 RBS：核糖体结合位点；Lac Z：β-半乳糖苷酶基因

Fig. 3 Diagnostic principles of NASBACC based on CRISPR-Cas9 system RBS: Ribosome binding site; Lac Z: β-galactosidase gene

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