Current Progress and Applications of CRISPR/Cas12a Gene Editing Technology in Plants

doi:10.13560/j.cnki.biotech.bull.1985.2024-1160

Abstract

Abstract:

‍The CRISPR/Cas system provides bacteria and archaea with adaptive immunity against viruses and plasmids by using crRNAs to guide the silencing of invading nucleic acids. The development and optimization of CRISPR/Cas system have provided a variety of gene editing tools for life science research, such as CRISPR/Cas9 genome editing technology, CRISPR/Cas12a genome editing technology, base editing, and prime editing. These tools are capable of precisely editing target genomes to generate various types of desired mutants, and show great application potential in functional genomics, construction of disease models, gene therapy and crop breeding. CRISPR/Cas12a genome editing technology is developed based on Class II type V CRISPR. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA. Besides, it presents distinct characteristics, such as the ability to utilize TTTV PAM, easy to engineer, and multiplex genome editing. Due to these advantages, CRISPR/Cas12a has been successfully applied in diverse species including animals and plants since the first report. Therefore, CRISPR/Cas12a will have a high prospect of providing important technical support for gene therapy and crop breeding. In this review, we provide a detailed introduction to the CRISPR/Cas12a genome editing technology, as well as the precise genome editing technologies developed based on CRISPR/Cas12a. We also focus on the strategies for optimizing CRISPR/Cas12a through improving editing efficiency, expanding the targeting scope, and enhancing specificity. Finally, we summarize the current applications of CRISPR/Cas12a in the creation of new plant germplasm. This review is aimed to facilitate the further application of CRISPR/Cas12a in crop improvement.

Key words: CRISPR/Cas12a, genome editing, optimization, genetic improvement

HUO Guan-zhong, ZHANG Xin-ru, TIAN Shi-jun, LI Jun. Current Progress and Applications of CRISPR/Cas12a Gene Editing Technology in Plants[J]. Biotechnology Bulletin, 2025, 41(6): 1-11.

Figures/Tables 4

References 74

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分类 Type	Cas蛋白 Cas protein	大小 Size （aa）	PAM类型 PAM type	RNA 组成 RNA requirement	多基因编辑 Multiple-gene editing	参考文献 Reference
Ⅱ	SpCas9	1 368	NGG	crRNA+tracrRNA	是	［6］
V-A	LbCas12a	1 223	TTTV	crRNA	是	［9］
	AsCas12a	1 307	TTV	crRNA	是
	FnCas12a	1 300	TTN	crRNA	是
V-B	AaCas12b	1 129	TTN	crRNA+tracrRNA	否	［10］
V-C	Cas12c	1 218	TN	crRNA+tracrRNA	否	［11］
V-F	SpCas12f	497	TTN	crRNA+tracrRNA	否	［12］
V-I	Cas12i	1 093	TTN	crRNA	否	［13］
V-J	Cas12j	700-800	TBN	crRNA	否	［14］

分类 Type	Cas蛋白 Cas protein	大小 Size （aa）	PAM类型 PAM type	RNA 组成 RNA requirement	多基因编辑 Multiple-gene editing	参考文献 Reference
Ⅱ	SpCas9	1 368	NGG	crRNA+tracrRNA	是	［6］
V-A	LbCas12a	1 223	TTTV	crRNA	是	［9］
	AsCas12a	1 307	TTV	crRNA	是
	FnCas12a	1 300	TTN	crRNA	是
V-B	AaCas12b	1 129	TTN	crRNA+tracrRNA	否	［10］
V-C	Cas12c	1 218	TN	crRNA+tracrRNA	否	［11］
V-F	SpCas12f	497	TTN	crRNA+tracrRNA	否	［12］
V-I	Cas12i	1 093	TTN	crRNA	否	［13］
V-J	Cas12j	700-800	TBN	crRNA	否	［14］

物种 Species	Cas12a种类 Cas12a type	PAM 类型 PAM type	靶基因 Target gene	编辑类型 Editing type	表型 Phenotype	参考文献 References
水稻 Oryza sativa	Mb3Cas12a	TTTV TTV	OsGBSS1 OsD18	多基因敲除 Multiple knockout	直链淀粉减少；株高连续变异	［49］
	FnCas12a	TTN	OsDL	敲除Knockout	叶片下垂	［51］
	LbCas12a	TTTV	OsEPFL9	敲除Knockout	叶表面气孔密度降低	［60］
	LbCas12a	TTTV	OsROC5	插入Insertion	叶片卷曲	［61］
	LbCas12a	TTTV	OsALS	定点替换Replacement	抗除草剂	［62］
	LbCas12a	TTTV	OsD18	多基因敲除 Multiple knockout	株高连续变异	［20］
	LbCas12a	TTTV	OsMIR394	敲除Knockout	千粒重增加	［63］
玉米 Zea mays	LbCas12a	TTTV	ZmGl2	敲除Knockout	叶片表皮无蜡质	［64］
棉花 Gossypium hirsutum	LbCas12a	TTTV	GhCLA	敲除Knockout	植株白化	［55］
棉花 Gossypium hirsutum	LbCas12a	TTTV	GhPGF	敲除Knockout	种子不含棉酚	［65］
番茄 Solanum lycopersicum	Mb3Cas12a	TTV TTTV	SlMYBATV， SlGGP1，SlCYCB	多基因敲除 Multiple knockout	番茄红素含量增加	［49］
番茄 Solanum lycopersicum	LbCas12a	TTTV	SlHKT1；2	基因定点敲入 GT	提高耐盐性	［57］
柑橘 Citrus reticulata	LbCas12a	TTTV	CsLOB1	敲除Knockout	抗柑橘溃疡病	［56］
柑橘 Citrus reticulata	LbCas12a	TTTV	CsLOBP	敲除Knockout	抗柑橘溃疡病	［66］
小麦 Triticum aestivum	LbCas12a	TTTV	TaGW7-B1	多基因敲除 Multiple knockout	增加籽粒大小和重量	［53］
小麦 Triticum aestivum	LbCas12a	TTTV	TaMTL	敲除Knockout	结实率降低	［67］
杨树 Populus	LbCas12a	TTTV	Pt4CL1， PtPII， PtSVP	多基因敲除 Multiple knockout	-	［42］
大豆 Glycine max	LbCas12a	TTTV	GmFAD2	大片段删除 Segment deletions	-	［68］
大豆 Glycine max	LbCas12a	TTTV	GmFAD2	敲除Knockout	-	［69］
葡萄 Vitis vinifera L.	LbCas12a	TTTV	TMT1 DFR1	多基因敲除 Multiple knockout	糖积累减少；改变类黄酮积累	［70］
高羊茅 Festuca arundinacea	LbCas12a	TTTV	FaPDS	敲除Knockout	植株白化	［71］
芥菜 Brassica juncea	LbCas12a	TTTV	I型黑芥子酶基因家族	敲除Knockout	降低新鲜叶片刺激性气味	［72］

物种 Species	Cas12a种类 Cas12a type	PAM 类型 PAM type	靶基因 Target gene	编辑类型 Editing type	表型 Phenotype	参考文献 References
水稻 Oryza sativa	Mb3Cas12a	TTTV TTV	OsGBSS1 OsD18	多基因敲除 Multiple knockout	直链淀粉减少；株高连续变异	［49］
	FnCas12a	TTN	OsDL	敲除Knockout	叶片下垂	［51］
	LbCas12a	TTTV	OsEPFL9	敲除Knockout	叶表面气孔密度降低	［60］
	LbCas12a	TTTV	OsROC5	插入Insertion	叶片卷曲	［61］
	LbCas12a	TTTV	OsALS	定点替换Replacement	抗除草剂	［62］
	LbCas12a	TTTV	OsD18	多基因敲除 Multiple knockout	株高连续变异	［20］
	LbCas12a	TTTV	OsMIR394	敲除Knockout	千粒重增加	［63］
玉米 Zea mays	LbCas12a	TTTV	ZmGl2	敲除Knockout	叶片表皮无蜡质	［64］
棉花 Gossypium hirsutum	LbCas12a	TTTV	GhCLA	敲除Knockout	植株白化	［55］
棉花 Gossypium hirsutum	LbCas12a	TTTV	GhPGF	敲除Knockout	种子不含棉酚	［65］
番茄 Solanum lycopersicum	Mb3Cas12a	TTV TTTV	SlMYBATV， SlGGP1，SlCYCB	多基因敲除 Multiple knockout	番茄红素含量增加	［49］
番茄 Solanum lycopersicum	LbCas12a	TTTV	SlHKT1；2	基因定点敲入 GT	提高耐盐性	［57］
柑橘 Citrus reticulata	LbCas12a	TTTV	CsLOB1	敲除Knockout	抗柑橘溃疡病	［56］
柑橘 Citrus reticulata	LbCas12a	TTTV	CsLOBP	敲除Knockout	抗柑橘溃疡病	［66］
小麦 Triticum aestivum	LbCas12a	TTTV	TaGW7-B1	多基因敲除 Multiple knockout	增加籽粒大小和重量	［53］
小麦 Triticum aestivum	LbCas12a	TTTV	TaMTL	敲除Knockout	结实率降低	［67］
杨树 Populus	LbCas12a	TTTV	Pt4CL1， PtPII， PtSVP	多基因敲除 Multiple knockout	-	［42］
大豆 Glycine max	LbCas12a	TTTV	GmFAD2	大片段删除 Segment deletions	-	［68］
大豆 Glycine max	LbCas12a	TTTV	GmFAD2	敲除Knockout	-	［69］
葡萄 Vitis vinifera L.	LbCas12a	TTTV	TMT1 DFR1	多基因敲除 Multiple knockout	糖积累减少；改变类黄酮积累	［70］
高羊茅 Festuca arundinacea	LbCas12a	TTTV	FaPDS	敲除Knockout	植株白化	［71］
芥菜 Brassica juncea	LbCas12a	TTTV	I型黑芥子酶基因家族	敲除Knockout	降低新鲜叶片刺激性气味	［72］

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