Establishment of CRISPR/CasX-based Genome Editing Technology in Rice

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

Abstract

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

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.

Figures/Tables 8

References 29

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	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%）