CRISPR-Cas12a基因编辑技术及其在农业生产中的应用

doi:10.3864/j.issn.0578-1752.2025.07.014

摘要/Abstract

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-Cas12a系统在分子检测领域的作用原理；从基因调控、碱基编辑、物质检测、疾病诊断和生物育种几个方面系统阐述了CRISPR-Cas12a在农业生产中的应用。随着引物编辑等更加安全的非DSB依赖技术的发展，CRISPR-Cas12a系统将在作物精准育种、畜禽遗传改良和临床快速检测等领域发挥更重要的作用，为应对全球粮食安全挑战和传染病防控提供创新性解决方案。

关键词: CRISPR-Cas12a, 基因编辑, 疾病诊断, 分子检测

Abstract:

The 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 and CRISPR-Cas9 was conducted across four dimensions: crRNA processing mechanisms, structural-functional features of Cas effectors, editing efficiency, and application scenarios. Additionally, the regulatory systems of CRISPR-dCas12a and CRISPR-dCas9 were evaluated regarding gene expression modulation, epigenetic editing, and base editing. The review also elucidated the molecular detection principles of CRISPR-Cas12a in targeting nucleic acids, proteins, and small molecules, as well as its agricultural applications in gene regulation, base editing, pathogen detection, disease diagnosis, and bio-breeding.With the emergence of safer non-DSB- dependent technologies such as prime editing, the CRISPR-Cas12a system was poised to play an increasingly vital role in crop precision breeding, livestock genetic improvement, and rapid clinical diagnostics. These advancemented promise innovative solutions to global food security challenges and infectious disease control, further cementing CRISPR-Cas12a as a cornerstone tool in agricultural biotechnology and molecular medicine.

Key words: CRISPR-Cas12a, gene editing, disease diagnosis, molecular detection

罗刚, 程依依, 杨雯, 肖怡梦, 杨铖熹. CRISPR-Cas12a基因编辑技术及其在农业生产中的应用[J]. 中国农业科学, 2025, 58(7): 1434-1450.

LUO Gang, CHENG YiYi, YANG Wen, XIAO YiMeng, YANG ChengXi. CRISPR-Cas12a Gene Editing Technology and Its Application in Agricultural Production[J]. Scientia Agricultura Sinica, 2025, 58(7): 1434-1450.

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核酸酶Nuclease	Cas9	Cas12a
类型Type	II	V
大小Size	~1000—1600aa	~1300aa
结构域Domain	RuvC和HNH	RuvC-Nuc
gRNA	crRNA和tracrRNA	crRNA
底物Substrate	dsDNA	ssDNA和dsDNA
PAMs	富含G G-rich region	富含T T-rich region
DSB	平末端Blunt end	黏性末端Sticky end
pre-crRNA加工 pre-crRNA processing	宿主RNase III和tracrRNA Host RNase III and tracrRNA	RNase活性RNase activity
种子序列 Seed sequence	PAM的3'端3' end of PAM	PAM的5'端5' end of PAM
附带切割 Collateral cleavage	否No	是Yes
靶向序列长度 Target sequence length	20 nt	23—25 nt
缺失核苷酸 Deletion nucleotide	1—2 nt	3—30 nt
多位点编辑 Multiple editing	较难Harder	容易Easier
HDR效率 HDR efficiency	非常低Lower	相对高Higher
体外快速检测 Rapid detection in vitro	设计繁琐Cumbersome design	广泛应用Widespread use

名称Name	基因型Genotype	PAM(5' to 3')	参考序列Reference
AsCas12a	野生型Wild-type	GTTV, GCTV	[25]
AsCas12a	野生型Wild-type	TTTV	[26]
AsCas12a-RR	S542R/K607R	TYCV	[23]
AsCas12a-RVR	S542R/K548V/N552R	TATV	[23]
enAsCas12a	E174R/S542R/K548R	TTYN, VTTV, TRTV	[27]
enAsCas12a-HF	E174R/N282A/S542R/K548R	TTYN, VTTV, TRTV	[27]
AsCas12a-Plus	R951K,R955A, E174R	ATTA, CTTA, GTTA, TCTA	[28]
LbCas12a	野生型Wild-type	TTTV	[26]
LbCas12a-RR	G532R/K595R	TYCV	[23]
LbCas12a-RVR	G532R/K538V/Y542R	TATV	[23]
LbCas12a-RVRR	G532R/K538V/Y542R/K595R	TNTN, TWCV, CYCV	[13]
impLbCas12a	D156R/G532R/K538V/Y542R/K595R	NTTV, TAYV, YYYV, TGTV	[13]
ttLbCas12a	D156R	TTTV	[29]
FnCas12a	野生型Wild-type	TTV, TTTV	[30]
FnCas12a-RR	N607R/K671R	TYCV, TCTV	[30]
FnCas12a-RVR	N607R/K613V/N617R	TWTV	[30]
FnCas12a-EP15	N607R/K613V/N617R/K180S/K660R/D616N	YN, TAC, CAA	[31]
FnCas12a-EP16	N607R/K613V/N617R/K180S/K660R	YN, TAC	[31]
MbCas12a	野生型Wild-type	TTV, TTTV	[30]
MbCas12a-RR	N576R/K637R	TYCV, TCTV	[30]
MbCas12a-RVR	N576R/K582V/N586R	TWTV	[30]
Mb2Cas12a	野生型Wild-type	VTTV	[11]
Mb2Cas12a-RVR	N563R/K569V/N573R	TATV	[11]
Mb2Cas12a-RVRR	N563R/K569V/N573R/K625R	NTTV, TATV, TYCV, CYCV	[11]
Mb3Cas12a	野生型Wild-type	TTTV, TTN	[32]
Mb3Cas12a-3Rv	D180R/N581R/K587R	NTTV, NTCV, TRTV	[33]
ErCas12a	野生型Wild-type	YTTN	[11]
ErCas12a-RR	D529R/K594R	TYCV	[11]
ErCas12a-RVR	D529R/K535V/N539R	TATV	[11]
CeCas12a	野生型Wild-type	TTTV	[34]
BsCas12a	野生型Wild-type	TTN	[35]
BsCas12a-3Rv	K155R/N512R/K518R	NTTV, NTCV, TRTV	[33]
PrCas12a-3Rv	E162R/N519R/K525R	NTTV, NTCV, TRTV	[33]
ArCas12a	野生型Wild-type	TTN	[35]
PrCas12a	野生型Wild-type	TTN	[35]
HkCas12a	野生型Wild-type	YYN	[35]