CRISPR-Cas12a基因编辑技术及其在农业生产中的应用  

CRISPR-Cas12a Gene Editing Technology and Its Application in Agricultural Production

作　　者：罗刚 程依依 杨雯 肖怡梦 杨铖熹 LUO Gang;CHENG YiYi;YANG Wen;XIAO YiMeng;YANG ChengXi(Jiangsu Key Laboratory of Sericultural and Animal Biotechnology/School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang 212100,Jiangsu;Key Laboratory of Silkworm and Mulberry Genetic Improvement,Ministry of Agriculture and Rural Affairs/The Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang 212100,Jiangsu)

机构地区：[1]江苏科技大学生物技术学院/江苏省蚕桑与畜禽生物技术重点实验室,江苏镇江212100 [2]农业农村部蚕桑遗传改良重点实验室/中国农业科学院桑蚕科学研究中心,江苏镇江212100

出　　处：《中国农业科学》2025年第7期1434-1450,共17页Scientia Agricultura Sinica

基　　金：国家自然科学基金青年项目(32102542);江苏省研究生科研与实践创新计划项目(SJCX23_2243);大学生创新创业训练计划项目(202310289120Y)。

摘　　要：规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)相关蛋白(CRISPR-associated protein, Cas)基因编辑技术不仅引发了生命科学领域的革命性发展,还在农业领域带来了翻天覆地的变革。作为CRISPR基因编辑系统的重要分支,CRISPR-Cas12a系统凭借其独特的分子特征,在生物育种和疾病诊断领域展现出区别于经典CRISPR-Cas9系统的应用潜力。相较于II型Cas9系统,V型的Cas12a蛋白只有一个RuvC-Nuc核酸酶结构域,与Cas9蛋白的HNH-RuvC双核酸酶结构域形成鲜明对比。Cas12a切割靶DNA产生交错型双链断裂(double-stranded DNA break,DSB),并保留了CRISPR RNA(crRNA)和Cas12a的“R环(R-loop)”。R环的保留是CRISPR-Cas12a基因编辑系统拥有“附带切割”特性和开发核酸和小分子检测技术的结构基础。Cas12a识别富含胸腺嘧啶的原间隔区相邻基序(protospacer adjacent motif,PAM),是CRISPR-Cas基因编辑系统的有效补充,其依赖crRNA的自主加工能力显著区别于Cas9的反式激活CRISPR RNA(tracrRNA)依赖系统,在多基因同步编辑方面展现出更大的优势。Cas12a的以上特性使其在农作物遗传改良中取得突破性进展——已成功培育出抗病、高产的商业化农作物品种。在基础研究方向,失活的Cas12a(dCas12a)与转录调控或表观修饰元件融合表达,可实现不产生DSB的精准基因表达调控;其与等温扩增技术联用可实现疾病的可视化检测。本文从V型Cas蛋白的分类、Cas12a在细菌内的作用原理和Cas12a与crRNA复合体的功能结构域等方面系统地介绍了CRISPR-Cas12a基因编辑系统;从靶向RNA的加工原理、Cas效应蛋白的功能结构、基因编辑的效果和应用4个方面对比了CRISPR-Cas12a和CRISPR-Cas9两套基因编辑系统;从基因调控、表观修饰和碱基编辑3个方面阐述了CRISPR-dCas12a和CRISPR-dCas9激活和抑制系统;从核酸、蛋白质和小分子3个方面阐述了CRISPR-Cas12aThe Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)and CRISPR-associated protein(CRISPR-Cas)gene editing technology has not only revolutionized life sciences but also catalyzed transformative advancements in agriculture.As a critical branch of the CRISPR system,the CRISPR-Cas12a system exhibits unique molecular characteristics and distinct application potential in biological breeding and disease diagnosis compared to the classical CRISPR-Cas9 system.Unlike the Type II Cas9 system,the Type V Cas12a protein possesses a single RuvC-like nuclease domain,contrasting sharply with the dual HNH-RuvC nuclease domains of Cas9.Cas12a generates staggered double-strand breaks(DSBs)in target DNA while retaining the CRISPR RNA(crRNA)and Cas12a-formed"R-loop".The preservation of this R-loop constitutes the the structural basis for the collateral cleavage activity inherent to the CRISPR-Cas12a system,which underpins its utility in developing nucleic acid and small molecule detection technologies.Recognizing thymine-rich protospacer adjacent motifs(PAMs),CRISPR-Cas12a acts as a powerful complement to existing CRISPR-Cas systems.Its crRNA-dependent autonomous processing mechanism,distinct from the tracrRNA-dependent system of Cas9,offers superior advantages in multiplex gene editing.These features have driven breakthroughs in crop genetic improvement,including the successful development of disease-resistant and high-yield commercial crop varieties.In basic research,catalytically inactive Cas12a(dCas12a)fused with transcriptional regulators or epigenetic modifiers enables precise gene expression regulation without inducing DSBs.Furthermore,its integration with isothermal amplification techniques allows for visual disease detection.This review systematically introduced the CRISPR-Cas12a system from multiple perspectives:(1)classification of Type V Cas proteins,(2)mechanistic principles of Cas12a in bacterial immunity,and(3)functional domains of the Cas12a-crRNA complex.A comparative analysis between CRISPR-Cas12a an

关 键 词：CRISPR-Cas12a 基因编辑 疾病诊断 分子检测 

分 类 号：Q78[生物学—分子生物学]