CRISPR/Cas12a基因编辑技术在植物中的研究进展

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

摘要/Abstract

摘要：

CRISPR/Cas系统是细菌和古生菌长期进化形成的适应性免疫系统，科研人员对其进行开发和优化，建立了CRISPR/Cas9基因编辑技术、CRISPR/Cas12a基因编辑技术、单碱基编辑技术和引导编辑技术，为生命科学研究提供了众多基因编辑工具。这些基因编辑工具能够高效精准地编辑目的基因组，产生各种类型的突变体，在基因功能研究、疾病模型构建、基因治疗以及作物育种等领域展现出巨大的应用潜力。CRISPR/Cas12a作为Ⅱ类Ⅴ型成员，Cas12a（Cpf1）核酸酶分子量较小，识别TTTV PAM序列，仅依赖单个crRNA结构，且无需tracrRNA的加工，操作简单以及具有多基因编辑等优点备受关注。CRISPR/Cas12a基因编辑技术自开发以来，已经成功应用在各种动植物基因组编辑中，为基因治疗及作物育种等提供重要技术支撑。本文详细介绍了CRISPR/Cas12a基因编辑技术的原理及基于CRISPR/Cas12a开发的碱基编辑和引导编辑技术，重点阐述了通过提高基因编辑效率、扩展靶向编辑范围和提高特异性等方面对CRISPR/Cas12a进行优化的策略，着重总结了该技术在植物遗传改良方面的应用，以期为CRISPR/Cas12a在植物中的进一步应用提供参考。

关键词: CRISPR/Cas12a, 基因组编辑, 优化, 遗传改良

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

霍贯中, 张欣濡, 田士军, 李君. CRISPR/Cas12a基因编辑技术在植物中的研究进展[J]. 生物技术通报, 2025, 41(6): 1-11.

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.

图/表 4

参考文献 74

1	Pacesa M, Pelea O, Jinek M. Past, present, and future of CRISPR genome editing technologies [J]. Cell, 2024, 187(5): 1076-1100.
2	Li J, Yu X, Zhang C, et al. The application of CRISPR/Cas technologies to Brassica crops: current progress and future perspectives[J]. aBIOTECH, 2022, 3, 146-161.
3	Mohanraju P, Makarova KS, Zetsche B, et al. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems [J]. Science, 2016, 353(6299): aad5147.
4	李君, 张毅, 陈坤玲, 等. CRISPR/Cas系统: RNA靶向的基因组定向编辑新技术 [J]. 遗传, 2013, 35(11): 1265-1273.
	Li J, Zhang Y, Chen KL, et al. CRISPR/Cas: a novel way of RNA-guided genome editing [J]. Hereditas, 2013, 35(11): 1265-1273.
5	Chen KL, Wang YP, Zhang R, et al. CRISPR/cas genome editing and precision plant breeding in agriculture [J]. Annu Rev Plant Biol, 2019, 70: 667-697.
6	Li Y, Li WJ, Li J. The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science [J]. J Genet Genomics, 2021, 48(8): 661-670.
7	Li BS, Sun C, Li JY, et al. Targeted genome-modification tools and their advanced applications in crop breeding [J]. Nat Rev Genet, 2024, 25: 603-622.
8	Walton RT, Christie KA, Whittaker MN, et al. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants [J]. Science, 2020, 368(6488): 290-296.
9	Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system [J]. Cell, 2015, 163(3): 759-771.
10	Ming ML, Ren QR, Pan CT, et al. CRISPR-Cas12b enables efficient plant genome engineering [J]. Nat Plants, 2020, 6(3): 202-208.
11	Kurihara N, Nakagawa R, Hirano H, et al. Structure of the type V-C CRISPR-cas effector enzyme [J]. Mol Cell, 2022, 82(10): 1865-1877.e4.
12	Bigelyte G, Young JK, Karvelis T, et al. Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells [J]. Nat Commun, 2021, 12(1): 6191.
13	McGaw C, Garrity AJ, Munoz GZ, et al. Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing [J]. Nat Commun, 2022, 13(1): 2833.
14	Sun Y, Hu JJ, Hu ZC, et al. Engineer and split an efficient hypercompact CRISPR-CasΦ genome editor in plants [J]. Plant Commun, 2024, 5(7): 100881.
15	Swarts DC, van der Oost J, Jinek M. Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a [J]. Mol Cell, 2017, 66(2): 221-233.e4.
16	Fonfara I, Richter H, Bratovič M, et al. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA [J]. Nature, 2016, 532(7600): 517-521.
17	Zetsche B, Heidenreich M, Mohanraju P, et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array [J]. Nat Biotechnol, 2017, 35(1): 31-34.
18	Kim D, Kim J, Hur JK, et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells [J]. Nat Biotechnol, 2016, 34(8): 863-868.
19	Li SY, Zhang YX, Xia LQ, et al. CRISPR-Cas12a enables efficient biallelic gene targeting in rice [J]. Plant Biotechnol J, 2020, 18(6): 1351-1353.
20	Zhou JP, Liu GQ, Zhao YX, et al. An efficient CRISPR-Cas12a promoter editing system for crop improvement [J]. Nat Plants, 2023, 9(4): 588-604.
21	Li SY, Li JY, He YB, et al. Precise gene replacement in rice by RNA transcript-templated homologous recombination [J]. Nat Biotechnol, 2019, 37(4): 445-450.
22	Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors [J]. Nat Biotechnol, 2020, 38(7): 824-844.
23	Li XS, Wang Y, Liu YJ, et al. Base editing with a Cpf1-cytidine deaminase fusion [J]. Nat Biotechnol, 2018, 36(4): 324-327.
24	Wang X, Ding CF, Yu WX, et al. Cas12a base editors induce efficient and specific editing with low DNA damage response [J]. Cell Rep, 2020, 31(9): 107723.
25	Chen SY, Jia YQ, Liu ZQ, et al. Robustly improved base editing efficiency of Cpf1 base editor using optimized cytidine deaminases [J]. Cell Discov, 2020, 6: 62.
26	Chen FB, Lian M, Ma BX, et al. Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors [J]. Commun Biol, 2022, 5(1): 1163.
27	Kleinstiver BP, Sousa AA, Walton RT, et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing [J]. Nat Biotechnol, 2019, 37(3): 276-282.
28	Gaillochet C, Peña Fernández A, Goossens V, et al. Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform [J]. Genome Biol, 2023, 24(1): 6.
29	王敬文, 严芳, 柳浪, 等. 水稻CRISPR/Cas12a系统的优化及其介导的腺嘌呤碱基编辑器的建立 [J]. 生物技术通报, 2021, 37(6): 279-285.
	Wang JW, Yan F, Liu L, et al. Optimization of CRISPR/Cas12a system and development of it mediated adenine base editor in rice [J]. Biol Bull, 2021, 37(6): 279-285.
30	Cheng YH, Zhang YX, Li G, et al. CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice [J]. Plant Commun, 2023, 4(4): 100601.
31	Wang GY, Xu ZP, Wang FQ, et al. Development of an efficient and precise adenine base editor (ABE) with expanded target range in allotetraploid cotton (Gossypium hirsutum) [J]. BMC Biol, 2022, 20(1): 45.
32	Liang RH, He ZX, Zhao KT, et al. Prime editing using CRISPR-Cas12a and circular RNAs in human cells [J]. Nat Biotechnol, 2024, 42(12): 1867-1875.
33	Wei JJ, Liu JT, Wang ZW, et al. Engineering of a high-fidelity Cas12a nuclease variant capable of allele-specific editing [J]. PLoS Biol, 2024, 22(6): e3002680.
34	Zhang YX, Ren QR, Tang X, et al. Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems [J]. Nat Commun, 2021, 12(1): 1944.
35	Li JH, Liang QC, Zhou HP, et al. Profiling the impact of the promoters on CRISPR-Cas12a system in human cells [J]. Cell Mol Biol Lett, 2023, 28(1): 41.
36	Zhang XH, Xu LP, Fan RH, et al. Genetic editing and interrogation with Cpf1 and caged truncated pre-tRNA-like crRNA in mammalian cells [J]. Cell Discov, 2018, 4: 36.
37	Park HM, Liu H, Wu J, et al. Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo [J]. Nat Commun, 2018, 9(1): 3313.
38	Li B, Zhao WY, Luo X, et al. Engineering CRISPR-Cpf1 crRNAs and mRNAs to maximize genome editing efficiency [J]. Nat Biomed Eng, 2017, 1(5): 0066.
39	Moreno-Mateos MA, Fernandez JP, Rouet R, et al. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing [J]. Nat Commun, 2017, 8(1): 2024.
40	Malzahn AA, Tang X, Lee K, et al. Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis [J]. BMC Biol, 2019, 17(1): 9.
41	Schindele P, Puchta H. Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing [J]. Plant Biotechnol J, 2020, 18(5): 1118-1120.
42	Zhang LY, Li G, Zhang YX, et al. Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants [J]. Genome Biol, 2023, 24(1): 102.
43	Xin CP, Qiao DX, Wang JY, et al. Enhanced editing efficiency in Arabidopsis with a LbCas12a variant harboring D156R and E795L mutations [J]. aBIOTECH, 2024, 5(2): 117-126.
44	Riesenberg S, Maricic T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells [J]. Nat Commun, 2018, 9(1): 2164.
45	Ma XJ, Chen X, Jin Y, et al. Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells [J]. Nat Commun, 2018, 9(1): 1303.
46	Zhang QW, Yin KQ, Liu GW, et al. Fusing T5 exonuclease with Cas9 and Cas12a increases the frequency and size of deletion at target sites [J]. Sci China Life Sci, 2020, 63(12): 1918-1927.
47	Gao LY, Cox DBT, Yan WX, et al. Engineered Cpf1 variants with altered PAM specificities [J]. Nat Biotechnol, 2017, 35(8): 789-792.
48	Hui FJ, Tang X, Li B, et al. Robust CRISPR/Mb2Cas12a genome editing tools in cotton plants [J]. iMETA, 2024, 3(3): e209.
49	Liu SS, He Y, Fan TT, et al. PAM-relaxed and temperature-tolerant CRISPR-Mb3Cas12a single transcript unit systems for efficient singular and multiplexed genome editing in rice, maize, and tomato [J]. Plant Biotechnol J, 2025, 23(1): 156-173.
50	Li SY, Zhang X, Wang WS, et al. Expanding the scope of CRISPR/Cpf1-mediated genome editing in rice [J]. Mol Plant, 2018, 11(7): 995-998.
51	Endo A, Masafumi M, Kaya H, et al. Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida [J]. Sci Rep, 2016, 6: 38169.
52	Dong SJ, Qin YL, Vakulskas CA, et al. Efficient targeted mutagenesis mediated by CRISPR-Cas12a ribonucleoprotein complexes in maize [J]. Front Genome Ed, 2021, 3: 670529.
53	Wang W, Tian B, Pan QL, et al. Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases [J]. Plant Biotechnol J, 2021, 19(12): 2428-2441.
54	Banakar R, Schubert M, Kurgan G, et al. Efficiency, specificity and temperature sensitivity of Cas9 and Cas12a RNPs for DNA-free genome editing in plants [J]. Front Genome Ed, 2022, 3: 760820.
55	Li B, Rui HP, Li YJ, et al. Robust CRISPR/Cpf1 (Cas12a)-mediated genome editing in allotetraploid cotton (Gossypium hirsutum) [J]. Plant Biotechnol J, 2019, 17(10): 1862-1864.
56	Su H, Wang YC, Xu J, et al. Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation [J]. Nat Commun, 2023, 14(1): 3957.
57	Vu TV, Sivankalyani V, Kim EJ, et al. Highly efficient homology-directed repair using CRISPR/Cpf1-geminiviral replicon in tomato [J]. Plant Biotechnol J, 2020, 18(10): 2133-2143.
58	Ferenczi A, Pyott DE, Xipnitou A, et al. Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA [J]. Proc Natl Acad Sci USA, 2017, 114(51): 13567-13572.
59	Pu XJ, Liu LN, Li P, et al. A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens [J]. Plant J, 2019, 100(4): 863-872.
60	Yin XJ, Biswal AK, Dionora J, et al. CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice [J]. Plant Cell Rep, 2017, 36(5): 745-757.
61	Tang X, Lowder LG, Zhang T, et al. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants [J]. Nat Plants, 2017, 3: 17018.
62	Li SY, Li JY, Zhang JH, et al. Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice [J]. J Exp Bot, 2018, 69(20): 4715-4721.
63	Zheng XL, Tang X, Wu YC, et al. An efficient CRISPR-Cas12a-mediated microRNA knockout strategy in plants [J]. Plant Biotechnol J, 2025, 23(1): 128-140.
64	Lee K, Zhang YX, Kleinstiver BP, et al. Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize [J]. Plant Biotechnol J, 2019, 17(2): 362-372.
65	Li B, Liang SJ, Alariqi M, et al. The application of temperature sensitivity CRISPR/LbCpf1 (LbCas12a) mediated genome editing in allotetraploid cotton (G. hirsutum) and creation of nontransgenic, gossypol-free cotton [J]. Plant Biotechnol J, 2021, 19(2): 221-223.
66	Jia HG, Orbović V, Wang N. CRISPR-LbCas12a-mediated modification of Citrus [J]. Plant Biotechnol J, 2019, 17(10): 1928-1937.
67	Liu HY, Wang K, Jia ZM, et al. Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system [J]. J Exp Bot, 2020, 71(4): 1337-1349.
68	Duan KX, Cheng YY, Ji J, et al. Large chromosomal segment deletions by CRISPR/LbCpf1-mediated multiplex gene editing in soybean [J]. J Integr Plant Biol, 2021, 63(9): 1620-1631.
69	Kim H, Kim ST, Ryu J, et al. CRISPR/Cpf1-mediated DNA-free plant genome editing [J]. Nat Commun, 2017, 8: 14406.
70	Ren C, Gathunga EK, Li X, et al. Efficient genome editing in grapevine using CRISPR/LbCas12a system [J]. Mol Hortic, 2023, 3(1): 21.
71	Zhang L, Wang T, Wang GY, et al. Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses [J]. J Integr Plant Biol, 2021, 63(8): 1410-1415.
72	Karlson D, Mojica JP, Poorten TJ, et al. Targeted mutagenesis of the multicopy Myrosinase gene family in allotetraploid Brassica juncea reduces pungency in fresh leaves across environments [J]. Plants, 2022, 11(19): 2494.
73	Tang X, Zhang Y. Beyond knockouts: fine-tuning regulation of gene expression in plants with CRISPR-Cas-based promoter editing [J]. New Phytol, 2023, 239(3): 868-874.
74	Jiang K, Yan Z, Di Bernardo M, et al. Rapid in silico directed evolution by a protein language model with EVOLVEpro [J]. Science, 2025, 387(6732): eadr6006.

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］

[1]	高畅, 庄添驰, 李宁, 刘云, 顾鹏飞, 赵昕怡, 季明辉. RPA-CRISPR/Cas12a结合重力驱动微流控芯片的MTB快检方法的建立[J]. 生物技术通报, 2025, 41(5): 62-69.
[2]	刘爽, 江洲, 赵帅, 赵雷真, 黄峰, 周佳, 屈建航. 一株产蛋白酶细菌的筛选、鉴定及发酵工艺优化[J]. 生物技术通报, 2025, 41(4): 335-344.
[3]	陈海敏, 孙菲, 袁源, 吴佳雯, 江红, 周剑. 紫外诱变选育巴弗洛霉素A1高产菌株及其培养基优化[J]. 生物技术通报, 2025, 41(3): 51-61.
[4]	宋倩娜, 段永红, 冯瑞云. CRISPR/Cas9介导的高效四倍体马铃薯试管薯基因编辑体系的建立[J]. 生物技术通报, 2024, 40(9): 33-41.
[5]	张梦菲, 余炼, 李菲, 李喆, 苏芯莹, 蓝彩碧, 朱虎, 秦莹. 响应面法优化暹罗芽孢杆菌产大环内酯的发酵培养条件[J]. 生物技术通报, 2024, 40(8): 299-308.
[6]	侯文婷, 孙琳, 张艳军, 董合忠. 基因编辑技术在棉花种质创新和遗传改良中的应用[J]. 生物技术通报, 2024, 40(7): 68-77.
[7]	陈墨岩, 祝诚. 基于CRISPR/Cas12a的生物传感平台的机制研究及应用[J]. 生物技术通报, 2024, 40(7): 90-98.
[8]	杨鹭, 袁源, 方志锴, 林如, 江红, 周剑. 一株链霉菌的鉴定及其产格尔德霉素的发酵工艺研究[J]. 生物技术通报, 2024, 40(6): 299-309.
[9]	茹扎·也里扎提, 杨宇. 毕赤酵母中外源蛋白表达量的提升策略[J]. 生物技术通报, 2024, 40(3): 118-134.
[10]	李蕊蕊, 那道远, 王洪林, 赵亮, 谭文松, 叶倩. 重组腺相关病毒瞬时表达系统的理性设计及其高效生产工艺的建立[J]. 生物技术通报, 2024, 40(12): 61-71.
[11]	杨帅朋, 屈子啸, 朱向星, 唐冬生. DNA碱基编辑技术的研究进展及在猪基因修饰中的应用[J]. 生物技术通报, 2024, 40(1): 127-144.
[12]	江海溶, 崔若琪, 王玥, 白淼, 张明露, 任连海. NH₃和H₂S降解功能菌的分离鉴定及降解特性研究[J]. 生物技术通报, 2023, 39(9): 246-254.
[13]	李雪琪, 张素杰, 于曼, 黄金光, 周焕斌. 基于CRISPR/CasX介导的水稻基因组编辑技术的建立[J]. 生物技术通报, 2023, 39(9): 40-48.
[14]	张岳一, 兰社益, 裴海闰, 封棣. 多菌种联用发酵燕麦麸皮工艺优化及发用功效评价[J]. 生物技术通报, 2023, 39(9): 58-70.
[15]	赵光绪, 杨合同, 邵晓波, 崔志豪, 刘红光, 张杰. 一株高效溶磷产红青霉培养条件优化及其溶磷特性[J]. 生物技术通报, 2023, 39(9): 71-83.