基于CRISPR/CasX介导的水稻基因组编辑技术的建立

doi:10.13560/j.cnki.biotech.bull.1985.2023-0413

摘要/Abstract

摘要：

Cas蛋白作为核酸酶发挥其切割活性需要识别特定的PAM序列，如SpCas9识别NGG PAM位点，LbCas12a识别TTTV PAM。已挖掘到新的能够识别TTCN PAM序列的蛋白—CasX蛋白，扩展了基因组编辑技术的编辑范围。本研究利用CasX的两个衍生型蛋白PlmCasX和DpbCasX，建立基于CRISPR/CasX介导的水稻基因编辑系统。通过PEG介导的水稻原生质体瞬时表达分析其编辑活性发现，PlmCasX和DpbCasX两个蛋白能够对水稻内源基因OsCPK16实现有效编辑。后通过水稻稳定遗传转化进一步验证，在TTCA PAM识别位点，DpbCasX蛋白对水稻内源基因OsCPK21的编辑效率为17.5%，PlmCasX蛋白对水稻内源基因OsCPK21的编辑效率为66.07%；在TTCG PAM识别位点，PlmCasX蛋白对OsCPK4的编辑效率为23.21%，而DpbCasX蛋白不能实现有效的基因编辑。并且基于MIDAS方法对PlmCasX蛋白的优化并不能提高其编辑活性。本研究证明了CRISPR/CasX系统在水稻中具有编辑活性，且其能识别TTCR PAM这一特性，扩大了基因编辑技术在水稻中的应用范围。

关键词: 水稻, 基因组编辑, CRISPR, CasX

Abstract:

As a nuclease, the Cas protein needs to recognize a specific protospacer adjacent motifs（PAM）sequence to exert its cleavage activity, for example SpCas9 recognizes the NGG PAM and LbCas12a recognizes the TTTV PAM. CasX protein, a new protein that can recognize the TTCN PAM sequence was discovered，which expands the editing range of genome editing technology. Using PlmCasX and DpbCasX derived from CasX, CRISPR/CasX-mediated rice gene editing system was established. The editing efficiency through transient expression of rice protoplasts mediated with PEG was analysed, and the results showed that the endogenous OsCPK16 was effectively edited by PlmCasX and DpbCasX. The subsequent rice stable genetic transformation results indicated DpbCasX showed 17.5% editing efficiency in OsCPK21 at TTCA PAM sites, while PlmCasX showed higher editing efficiency in OsCPK21 at TTCA PAM sites and in OsCPK4 at TTCG PAM sites, with editing efficiency of 66.07% and 23.21%, respectively. And the optimization of PlmCasX protein based on the MIDAS method could not improve its editing activity. The study proved that the CRISPR/CasX system has editing activity in rice, and it can recognize the characteristic of TTCR PAM, expanding the application range of gene editing technology in rice.

Key words: Oryza sativa L., genome editing, CRISPR, CasX

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.

LI Xue-qi, ZHANG Su-jie, YU Man, HUANG Jin-guang, ZHOU Huan-bin. Establishment of CRISPR/CasX-based Genome Editing Technology in Rice[J]. Biotechnology Bulletin, 2023, 39(9): 40-48.

图/表 8

参考文献 29

[1]	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. doi: 10.1146/annurev-arplant-050718-100049 pmid: 30835493
[2]	Ceasar SA, Rajan V, Prykhozhij SV, et al. Insert, remove or replace: a highly advanced genome editing system using CRISPR/Cas9[J]. Biochim Biophys Acta, 2016, 1863(9): 2333-2344. doi: 10.1016/j.bbamcr.2016.06.009 pmid: 27350235
[3]	Hille F, Richter H, Wong SP, et al. The biology of CRISPR-cas: backward and forward[J]. Cell, 2018, 172(6): 1239-1259. doi: S0092-8674(17)31383-1 pmid: 29522745
[4]	Shan QW, Wang YP, Li J, et al. Targeted genome modification of crop plants using a CRISPR-Cas system[J]. Nat Biotechnol, 2013, 31(8): 686-688. doi: 10.1038/nbt.2650 pmid: 23929338
[5]	Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096): 816-821. doi: 10.1126/science.1225829 pmid: 22745249
[6]	Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III[J]. Nature, 2011, 471(7340): 602-607. doi: 10.1038/nature09886
[7]	Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121): 819-823. doi: 10.1126/science.1231143 pmid: 23287718
[8]	Wang MX, Xu ZY, Gosavi G, et al. Targeted base editing in rice with CRISPR/ScCas9 system[J]. Plant Biotechnol J, 2020, 18(8): 1645-1647. doi: 10.1111/pbi.13330 pmid: 31916673
[9]	Hua K, Tao XP, Zhu JK. Expanding the base editing scope in rice by using Cas9 variants[J]. Plant Biotechnol J, 2019, 17(2): 499-504. doi: 10.1111/pbi.12993 pmid: 30051586
[10]	Steinert J, Schiml S, Fauser F, et al. Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus[J]. Plant J, 2015, 84(6): 1295-1305. doi: 10.1111/tpj.2015.84.issue-6 URL
[11]	Hu JH, Miller SM, Geurts MH, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity[J]. Nature, 2018, 556(7699): 57-63. doi: 10.1038/nature26155 URL
[12]	Nishimasu H, Shi X, Ishiguro S, et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space[J]. Science, 2018, 361(6408): 1259-1262. doi: 10.1126/science.aas9129 pmid: 30166441
[13]	Ren B, Liu L, Li SF, et al. Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice[J]. Mol Plant, 2019, 12(7): 1015-1026. doi: S1674-2052(19)30123-6 pmid: 30928635
[14]	Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J]. Proc Natl Acad Sci USA, 2012, 109(39): E2579-E2586.
[15]	Li XS, Wang Y, Liu YJ, et al. Base editing with a Cpf1-cytidine deaminase fusion[J]. Nat Biotechnol, 2018, 36(4): 324-327. doi: 10.1038/nbt.4102 pmid: 29553573
[16]	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. doi: 10.1016/j.cell.2015.09.038 pmid: 26422227
[17]	Burstein D, Harrington LB, Strutt SC, et al. New CRISPR-cas systems from uncultivated microbes[J]. Nature, 2017, 542(7640): 237-241. doi: 10.1038/nature21059
[18]	Cao CC, Yao L, Li AL, et al. A CRISPR/dCasX-mediated transcriptional programming system for inhibiting the progression of bladder cancer cells by repressing c-MYC or activating TP53[J]. Clin Transl Med, 2021, 11(9): e537. doi: 10.1002/ctm2.537 pmid: 34586743
[19]	Roberson EDO. A catalog of CasX genome editing sites in common model organisms[J]. BMC Genomics, 2019, 20(1): 528. doi: 10.1186/s12864-019-5924-6 pmid: 31248380
[20]	Liu JJ, Orlova N, Oakes BL, et al. CasX enzymes comprise a distinct family of RNA-guided genome editors[J]. Nature, 2019, 566(7743): 218-223. doi: 10.1038/s41586-019-0908-x
[21]	Yang H, Patel DJ. CasX: a new and small CRISPR gene-editing protein[J]. Cell Res, 2019, 29(5): 345-346. doi: 10.1038/s41422-019-0165-4 pmid: 30992542
[22]	Chen YC, Hu YP, Wang XG, et al. Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing[J]. Innovation(Camb), 2022, 3(4): 100264.
[23]	Zhou HB, Liu B, Weeks DP, et al. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice[J]. Nucleic Acids Res, 2014, 42(17): 10903-10914. doi: 10.1093/nar/gku806 pmid: 25200087
[24]	李超. 植物新型碱基编辑技术的开发及应用研究[D]. 北京: 中国科学院大学, 2020.
	Li C. Studies on developing new programmable base editors in plants and their applications[D]. Beijing: University of Chinese Academy of Sciences, 2020.
[25]	Hiei Y, Ohta S, Komari T, et al. Efficient transformation of rice(Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA[J]. Plant J, 1994, 6(2): 271-282. doi: 10.1046/j.1365-313x.1994.6020271.x pmid: 7920717
[26]	Nguyen VC, Nguyen VK, Singh CH, et al. Fast recovery of transgenic submergence tolerant rice cultivars of North-East India by early co-cultivation of Agrobacterium with pre-cultured callus[J]. Physiol Mol Biol Plants, 2017, 23(1): 115-123. doi: 10.1007/s12298-016-0398-3 URL
[27]	Tsuchida CA, Zhang SY, Doost MS, et al. Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity[J]. Mol Cell, 2022, 82(6): 1199-1209.e6. doi: 10.1016/j.molcel.2022.02.002 URL
[28]	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. doi: 10.13560/j.cnki.biotech.bull.1985.2021-0231
	Wang JW, Yan F, Liu L, et al. Optimization of CRISPR/Cas12a system and development of itmediated adenine base editor in rice[J]. Biotechnol Bull, 2021, 37(6): 279-285.
[29]	Roth MO, Li H. X marks the spot: mining the gold in CasX for gene editing[J]. Mol Cell, 2022, 82(6): 1083-1085. doi: 10.1016/j.molcel.2022.02.024 URL

	名称Name	抗性/种Resistance/Species	来源Source
质粒Plasmid	pUC57:PlmCasX	Amp^R	Synthetic
	pUC57:DpbCasX	Amp^R	Synthetic
	pUC57:PlmCasX-V1	Amp^R	Synthetic
	pHZ38	Kan^R	This study
	pENTR4:gRNA41	Kan^R	Lab stock
	pUC19:PlmCasX	Amp^R	This study
	pUC19:DpbCasX	Amp^R	This study
	pUC19:PlmCasX-V1	Amp^R	This study
	pUbi:PlmCasX	Kan^R	This study
	pUbi:DpbCasX	Kan^R	This study
	pUbi:PlmCasX-V1	Kan^R	This study
菌种Strain	JM109	Escherichia coli	Bought from TSINGKE
	DB3.1	Escherichia coli	Bought from TSINGKE
	EHA105	Agrobacterium tumefaciens	Bought from TSINGKE

	名称Name	抗性/种Resistance/Species	来源Source
质粒Plasmid	pUC57:PlmCasX	Amp^R	Synthetic
	pUC57:DpbCasX	Amp^R	Synthetic
	pUC57:PlmCasX-V1	Amp^R	Synthetic
	pHZ38	Kan^R	This study
	pENTR4:gRNA41	Kan^R	Lab stock
	pUC19:PlmCasX	Amp^R	This study
	pUC19:DpbCasX	Amp^R	This study
	pUC19:PlmCasX-V1	Amp^R	This study
	pUbi:PlmCasX	Kan^R	This study
	pUbi:DpbCasX	Kan^R	This study
	pUbi:PlmCasX-V1	Kan^R	This study
菌种Strain	JM109	Escherichia coli	Bought from TSINGKE
	DB3.1	Escherichia coli	Bought from TSINGKE
	EHA105	Agrobacterium tumefaciens	Bought from TSINGKE

引物名称Primer name	引物序列Primer sequence（5'-3'）
pUbi-PlmCasX-F1	ATTCCCGGGTACCGGATCCGCCACCATGGCCCCTAAAAAGAAGAGA
pUbi-PlmCasX-R1	GAGCGTTTTCATTGGCCCGCGCTTACCAGCTTTTTTTGTGTTCGAGTCT
pUbi-PlmCasX-F2	AGACTCGAACACAAAAAAAGCTGGTAAGCGCGGGCCAATGAAAACGCTC
pUbi-PlmCasX-R2	ACGCGGCCATTGGCCAGCCTGAGCGATCCGGTTTCCAAGGACAACAAAT
pUbi-PlmCasX-F3	ATTTGTTGTCCTTGGAAACCGGATCGCTCAGGCTGGCCAATGGCCGCGT
pUbi-PlmCasX-R3	CCCGCAGTTGCTACACGTCCTAGATGTATATTGTGCCAATGTCTTTGAC
pUbi-PlmCasX-F4	GTCAAAGACATTGGCACAATATACATCTAGGACGTGTAGCAACTGCGGG
pUbi-PlmCasX-R4	CGATCGGGGAAATTCGAGCTCACTAGTTCAGACCTTGCGCTTCTT
fgOsCPK16-F2	AAAGCTTCAGCACTTCAGGAGCCA
fgOsCPK16-R2	GGCCTGGCTCCTGAAGTGCTGAAG
fgOsCPK11-F2	AAAGCCACAAGCTCGTTGAAGCTT
fgOsCPK11-R2	GGCCAAGCTTCAACGAGCTTGTGG
fgOsCPK21-F1	AAAGACAGGGAGCCATTGCCGAGG
fgOsCPK21-R1	GGCCCCTCGGCAATGGCTCCCTGT
fgOsCPK23-F2	AAAGCGAACTGTCCCTGTCCAAGC
fgOsCPK23-R2	GGCCGCTTGGACAGGGACAGTTCG
fgOsCPK30-F3	AAAGGATCCACGGATGACCTGCAG
fgOsCPK30-R3	GGCCCTGCAGGTCATCCGTGGATC
fgOsCPK4-F2	AAGCCCTGTTGCTGTGGAGGATG
fgOsCPK4-R2	GCCCATCCTCCACAGCAACAGGG
fgOsCPK14-F1	AAAGTCCCCACCTCCCCTTTATCG
fgOsCPK14-R1	GGCCCGATAAAGGGGAGGTGGGGA
fgOsCPK27-F1	AAAGTGGCGAAGTTGCGGCGCGGG
fgOsCPK27-R1	GGCCCCCGCGCCGCAACTTCGCCA
OsCPK16-HF1	ggagtgagtacggtgtgcGTAAGACACCTCTTCACATTTCCC
OsCPK16-HR1	gagttggatgctggatggAAAAGGAGGAACTCCACATAACA
OsCPK23-HF2	ggagtgagtacggtgtgcCCTGTCCTTGGATACAAGACT
OsCPK23-HR2	gagttggatgctggatggGGTGCATTATCTGGATTTCAC
OsCPK30-HF2	ggagtgagtacggtgtgcATTGTGGCACTCGCTTTTTATC
OsCPK30-HR2	gagttggatgctggatggGAATAGTCATTTACAGACCAGAT
OsCPK27-HF1	ggagtgagtacggtgtgcTGTTGTGTGAAGAGAAGGTG
OsCPK27-HR1	gagttggatgctggatggAGGGGAGCACGACGGGGATGGA
OsCPK11-HF1	ggagtgagtacggtgtgcGCTTTTACATATTTTGAC
OsCPK11-HR1	gagttggatgctggatggTCTTGGATATTGTATAGTG
OsCPK21-HF1	ggagtgagtacggtgtgcGACAACGACGAGAAGATC
OsCPK21-HR1	gagttggatgctggatggTTAAGTTGTTTACTTACCAAG
OsCPK4-HF2	ggagtgagtacggtgtgcTGTGATTGAAGGCGATTT
OsCPK4-HR2	gagttggatgctggatggGAGTCATCTTCAAACGCA
OsCPK14-HF1	ggagtgagtacggtgtgcGCAACCGAACCCAAACCC
OsCPK14-HR1	gagttggatgctggatggGGGCAGCAGTTGCCCATG

引物名称Primer name	引物序列Primer sequence（5'-3'）
pUbi-PlmCasX-F1	ATTCCCGGGTACCGGATCCGCCACCATGGCCCCTAAAAAGAAGAGA
pUbi-PlmCasX-R1	GAGCGTTTTCATTGGCCCGCGCTTACCAGCTTTTTTTGTGTTCGAGTCT
pUbi-PlmCasX-F2	AGACTCGAACACAAAAAAAGCTGGTAAGCGCGGGCCAATGAAAACGCTC
pUbi-PlmCasX-R2	ACGCGGCCATTGGCCAGCCTGAGCGATCCGGTTTCCAAGGACAACAAAT
pUbi-PlmCasX-F3	ATTTGTTGTCCTTGGAAACCGGATCGCTCAGGCTGGCCAATGGCCGCGT
pUbi-PlmCasX-R3	CCCGCAGTTGCTACACGTCCTAGATGTATATTGTGCCAATGTCTTTGAC
pUbi-PlmCasX-F4	GTCAAAGACATTGGCACAATATACATCTAGGACGTGTAGCAACTGCGGG
pUbi-PlmCasX-R4	CGATCGGGGAAATTCGAGCTCACTAGTTCAGACCTTGCGCTTCTT
fgOsCPK16-F2	AAAGCTTCAGCACTTCAGGAGCCA
fgOsCPK16-R2	GGCCTGGCTCCTGAAGTGCTGAAG
fgOsCPK11-F2	AAAGCCACAAGCTCGTTGAAGCTT
fgOsCPK11-R2	GGCCAAGCTTCAACGAGCTTGTGG
fgOsCPK21-F1	AAAGACAGGGAGCCATTGCCGAGG
fgOsCPK21-R1	GGCCCCTCGGCAATGGCTCCCTGT
fgOsCPK23-F2	AAAGCGAACTGTCCCTGTCCAAGC
fgOsCPK23-R2	GGCCGCTTGGACAGGGACAGTTCG
fgOsCPK30-F3	AAAGGATCCACGGATGACCTGCAG
fgOsCPK30-R3	GGCCCTGCAGGTCATCCGTGGATC
fgOsCPK4-F2	AAGCCCTGTTGCTGTGGAGGATG
fgOsCPK4-R2	GCCCATCCTCCACAGCAACAGGG
fgOsCPK14-F1	AAAGTCCCCACCTCCCCTTTATCG
fgOsCPK14-R1	GGCCCGATAAAGGGGAGGTGGGGA
fgOsCPK27-F1	AAAGTGGCGAAGTTGCGGCGCGGG
fgOsCPK27-R1	GGCCCCCGCGCCGCAACTTCGCCA
OsCPK16-HF1	ggagtgagtacggtgtgcGTAAGACACCTCTTCACATTTCCC
OsCPK16-HR1	gagttggatgctggatggAAAAGGAGGAACTCCACATAACA
OsCPK23-HF2	ggagtgagtacggtgtgcCCTGTCCTTGGATACAAGACT
OsCPK23-HR2	gagttggatgctggatggGGTGCATTATCTGGATTTCAC
OsCPK30-HF2	ggagtgagtacggtgtgcATTGTGGCACTCGCTTTTTATC
OsCPK30-HR2	gagttggatgctggatggGAATAGTCATTTACAGACCAGAT
OsCPK27-HF1	ggagtgagtacggtgtgcTGTTGTGTGAAGAGAAGGTG
OsCPK27-HR1	gagttggatgctggatggAGGGGAGCACGACGGGGATGGA
OsCPK11-HF1	ggagtgagtacggtgtgcGCTTTTACATATTTTGAC
OsCPK11-HR1	gagttggatgctggatggTCTTGGATATTGTATAGTG
OsCPK21-HF1	ggagtgagtacggtgtgcGACAACGACGAGAAGATC
OsCPK21-HR1	gagttggatgctggatggTTAAGTTGTTTACTTACCAAG
OsCPK4-HF2	ggagtgagtacggtgtgcTGTGATTGAAGGCGATTT
OsCPK4-HR2	gagttggatgctggatggGAGTCATCTTCAAACGCA
OsCPK14-HF1	ggagtgagtacggtgtgcGCAACCGAACCCAAACCC
OsCPK14-HR1	gagttggatgctggatggGGGCAGCAGTTGCCCATG

PAM序列 PAM seq	基因 Gene	工具载体 Tool carrier	突变效率 Mutation efficiency	单等位基因突变 Mono-allelic mutation	双等位基因突变 Bi-allelic mutation
TTCG	OsCPK4	DpbCasX	0/48（0.00%）	0/0（0.00%）	0/0（0.00%）
	OsCPK4	PlmCasX	13/56（23.21%）	9/13（69.23%）	4/13（30.77%）
	OsCPK30	DpbCasX	0/50（0.00%）	0/0（0.00%）	0/0（0.00%）
	OsCPK30	PlmCasX	0/43（0.00%）	0/0（0.00%）	0/0（0.00%）
TTCA	OsCPK21	DpbCasX	7/40（17.50%）	5/7（71.43%）	2/7（28.57%）
TTCA	OsCPK21	PlmCasX	37/56（66.07%）	22/37（59.46%）	15/37（40.54%）
TTCC	OsCPK23	DpbCasX	0/56（0.00%）	0/0（0.00%）	0/0（0.00%）
	OsCPK23	PlmCasX	0/40（0.00%）	0/0（0.00%）	0/0（0.00%）
	OsCPK14	DpbCasX	0/42（0.00%）	0/0（0.00%）	0/0（0.00%）
	OsCPK14	PlmCasX	0/45（0.00%）	0/0（0.00%）	0/0（0.00%）
TTCT	OsCPK27	DpbCasX	0/42（0.00%）	0/0（0.00%）	0/0（0.00%）
	OsCPK27	PlmCasX	0/45（0.00%）	0/0（0.00%）	0/0（0.00%）
	OsCPK11	DpbCasX	0/40（0.00%）	0/0（0.00%）	0/0（0.00%）
	OsCPK11	PlmCasX	0/56（0.00%）	0/0（0.00%）	0/0（0.00%）