基于CRISPR/LanCas9的水稻基因编辑系统的开发和优化

doi:10.13560/j.cnki.biotech.bull.1985.2024-0475

生物技术通报 ›› 2024, Vol. 40 ›› Issue (10): 233-242.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0475

基于CRISPR/LanCas9的水稻基因编辑系统的开发和优化

李欣格¹^,²(), 王美霞²^,³, 王晨阳², 马桂根²^,⁴, 周常勇³, 王亚南¹(), 周焕斌²^,⁴^,⁵()

1.河北农业大学植物保护学院，保定 071001
2.中国农业科学院植物保护所植物病虫害综合治理全国重点实验室，北京 100193
3.西南大学柑橘研究所国家柑橘工程研究中心，重庆 400712
4.农业农村部基因编辑创新利用重点实验室（海南），三亚 572024
5.农业农村部桂林作物有害生物科学观测实验站，桂林 541399

收稿日期:2024-05-21 出版日期:2024-10-26 发布日期:2024-11-20
通讯作者: 王亚南，女，博士，教授，研究方向：植物病毒分子检测、果蔬病毒与寄主互作研究；E-mail: wyn3215347@163.com；
周焕斌，男，博士，研究员，研究方向：水稻与白叶枯病原菌的分子互作、基因组编辑技术开发与应用；E-mail: hbzhou@ippcaas.cn
作者简介:李欣格，女，硕士研究生，研究方向：基因组编辑技术开发与应用；E-mail: xingell@hotmail.com
基金资助:
科技创新2030—重大项目(2023ZD0407405-03)

Development and Optimization of Genome Editing in Rice with CRISPR/LanCas9 System

LI Xin-ge¹^,²(), WANG Mei-xia²^,³, WANG Chen-yang², MA Gui-gen²^,⁴, ZHOU Chang-yong³, WANG Ya-nan¹(), ZHOU Huan-bin²^,⁴^,⁵()

1. College of Plant Protection, Hebei Agricultural University, Baoding 071001
2. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
3. National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712
4. Key Laboratory of Gene Editing Technologies（Hainan）, Ministry of Agricultural and Rural Affairs, Sanya 572024
5. Scientific Observing and Experimental Station of Crop Pests in Guilin, Ministry of Agriculture and Rural Affairs, Guilin 541399

Received:2024-05-21 Published:2024-10-26 Online:2024-11-20

摘要/Abstract

摘要：

【目的】在水稻中建立CRISPR/LanCas9 和 CRISPR/SLanCas9基因编辑系统，扩展CRISPR/Cas基因编辑工具箱。【方法】将来自动物乳酸杆菌KCTC 3501的LanCas9进行密码子优化，并且进一步通过蛋白融合重组LanCas9和SpCas9的活性结构域形成嵌合体SLanCas9，分别构建CRISPR/LanCas9和CRISPR/SLanCas9编辑工具；以OsWRKY45、OsCPK4、OsCPK6、OsCPK7、OsMPK8、OsGSK3、OsGSK4为靶基因，在NGG PAM或者NAG PAM设计20 nt或者24 nt的sgRNA，通过水稻遗传转化，检测分析其编辑效率。【结果】LanCas9在水稻中可以识别NGG PAM，结合20 nt的sgRNA编辑效率更高，对OsWRKY45基因的编辑效率为25.00%；融合的SLanCas9不仅能够识别NGG PAM，在NAG PAM位点也有一定的编辑效率，在不同PAM位点的编辑效率分别可达100%和39.58%。此外，SLanCas9还具有多重基因编辑能力，在NGG PAM位点的多重基因编辑效率高达74.07%。【结论】开发了具有自主知识产权的新型CRISPR/LanCas9和CRISPR/SLanCas9基因编辑技术。

关键词: CRISPR, LanCas9, SLanCas9, 基因编辑, 水稻

Abstract:

【Objective】 The objective of this study is to develop CRISPR/LanCas9 and CRISPR/SLanCas9 editing systems for rice and to expand the CRISPR/Cas gene-editing toolkit. 【Method】 The codon of LanCas9 from Lactobacillus subtilis KCTC 3501 was optimized, and further a chimeric SLanCas9 by fusing the active domains of LanCas9 and SpCas9 was generated. Thus CRISPR/LanCas9 and CRISPR/SLanCas9 editing systems for rice were constructed respectively. Using OsWRKY45, OsCPK4, OsCPK6, OsCPK7, OsMPK8, OsGSK3, and OsGSK4 as target genes, the 20 nt or 24 nt sgRNAs in NGG PAM or NAG PAM were designed, and their editing efficiency was analyzed through rice genetic transformation.【Result】 The LanCas9 efficiently identified NGG PAM and efficiency was higher when combined with a 20-nt sgRNA editor, resulting in an editing efficiency of 25.00% for the OsWRKY45 gene. Furthermore, the fused SLanCas9 not only showed the recognition of the NGG PAM and also demonstrated a certain level of editing efficiency at the NAG PAM site, achieving editing efficiencies of 100% and 39.58% respectively for different PAM sites. Additionally, SLanCas9 demonstrated multiple-gene editing capabilities with an impressive efficiency of up to 74.07% at NGG PAM sites. 【Conclusion】 This study has successfully developed novel CRISPR/LanCas9 and CRISPR/SLanCas9 gene editing technologies with independent intellectual property rights.

Key words: CRISPR, LanCas9, SLanCas9, genome editing, rice

李欣格, 王美霞, 王晨阳, 马桂根, 周常勇, 王亚南, 周焕斌. 基于CRISPR/LanCas9的水稻基因编辑系统的开发和优化[J]. 生物技术通报, 2024, 40(10): 233-242.

LI Xin-ge, WANG Mei-xia, WANG Chen-yang, MA Gui-gen, ZHOU Chang-yong, WANG Ya-nan, ZHOU Huan-bin. Development and Optimization of Genome Editing in Rice with CRISPR/LanCas9 System[J]. Biotechnology Bulletin, 2024, 40(10): 233-242.

图/表 12

参考文献 30

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引物名称Primer name	序列Sequence（5'-3'）	用途Application
OsMPK8-F1	TGGCGGTGGAGGAGAGGTGA	Amplify the OsMPK8 target site in this research
OsMPK8-R1	CAACCACCCATGACCATGAG	Amplify the OsMPK8 target site in this research
OsWRKY45-F1	TGCAGCCGCTCAAGGTGGAG	Amplify the OsWRKY45 target site in this research
OsWRKY45-R1	TCAAACCCATAATGTCGTCC	Amplify the OsWRKY45 target site in this research
OsCPK6-F1	AGAAACAGCATCTCTTGAGG	Amplify the OsCPK6 target site in this research
OsCPK6-R1	GCTACTTGTTGCTGTGCTTG	Amplify the OsCPK6 target site in this research
OsCPK4-F1	TTCTCATCCCACACTGCGAC	Amplify two OsCPK4 target sites in this research
OsCPK4-R1	CCGAAATGGAACTCCCGAAT	Amplify two OsCPK4 target sites in this research
OsCPK7-F1	CCTGGGAAGGATACAATTGT	Amplify the OsCPK7 target site in this research
OsCPK7-R1	TAGCTATCCCCCTGAACGTA	Amplify the OsCPK7 target site in this research
OsGSK3-F1	TGTTCTGCCTTGCCTCACTGG	Amplify the OsGSK3 target site in this research
OsGSK3-R1	ACACTGCAGAAGTTTACCTG	Amplify the OsGSK3 target site in this research
OsGSK4-F1	GGACAAAGAATGTCGCATGA	Amplify the OsGSK4 target site in this research
OsGSK4-R1	ACATGCACAAGACTAATACG	Amplify the OsGSK4 target site in this research
gOsMPK8-F1	tgttgCGGAGGAAGGTGAGAGGAGG	Target the OsMPK8（20-nt）target site
gOsMPK8-R1	aaacCCTCCTCTCACCTTCCTCCGc	Target the OsMPK8（20-nt）target site
gOsMPK8-F2	tgttgCAGACGGAGCAGCAGCAGCAGCAG	Target the OsMPK8（24-nt）target site
gOsMPK8-R2	aaacCTGCTGCTGCTGCTGCTCCGTCTGc	Target the OsMPK8（24-nt）target site
gOsWRKY45-F1	tgttgTGGAGCTACGACGCCGTCGC	Target the OsWRKY45（20-nt）target site
gOsWRKY45-R1	aaacGCGACGGCGTCGTAGCTCCAc	Target the OsWRKY45（20-nt）target site
gOsWRKY45-F2	tgttgGCACTGGAGCTACGACGCCGTCGC	Target the OsWRKY45（24-nt）target site
gOsWRKY45-R2	aaacGCGACGGCGTCGTAGCTCCAGTGCc	Target the OsWRKY45（24-nt）target site
gOsCPK6-F1	gtgtgATGGGCAACTACTACTCGTG	Target the OsCPK6 target site
gOsCPK6-R1	aaacCACGAGTAGTAGTTGCCCATc	Target the OsCPK6 target site
gOsCPK4-F1	tgttGCGGGAGCGGGAAGCGGCAG	Target the OsCPK4 T1 target site
gOsCPK4-R1	aaacCTGCCGCTTCCCGCTCCCGC	Target the OsCPK4 T1 target site
gOsCPK4-F2	gtgtgTCGCCGTCAAGCGCATCGAC	Target the OsCPK4 T2 target site
gOsCPK4-R2	aaacGTCGATGCGCTTGACGGCGAc	Target the OsCPK4 T2 target site
gOsCPK7-F1	tgttgTGCCAGAACGGGACTCTTGG	Target the OsCPK7 target site
gOsCPK7-R1	aaacCCAAGAGTCCCGTTCTGGCAc	Target the OsCPK7 target site
gOsGSK3-F1	gtgtGGAAGAATGGAGAACCTAAA	Target the OsGSK3 target site
gOsGSK3-R1	aaacTTTAGGTTCTCCATTCTTCC	Target the OsGSK3 target site
gOsGSK4-F1	tgttgAACCTGGAATACAACACCAA	Target the OsGSK4 target site
gOsGSK4-R1	aaacTTGGTGTTGTATTCCAGGTTc	Target the OsGSK4 target site
g4-F1	TAAGCTTGATATCGAATTCG	Amplify the PENTR4:gRNA4 vector to construct the PENTR4:gRNA34 plasmid
g4-R1	TAGCCAACACAAGCGGCAGC
g34-F1	CGCTGCCGCTTGTGTTGGCTAGGATCCATCGCAGTCAGCG	Amplify the sgRNA fragments to construct the PENTR4:gRNA34 plasmid
g34-R1	CGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAG

引物名称Primer name	序列Sequence（5'-3'）	用途Application
OsMPK8-F1	TGGCGGTGGAGGAGAGGTGA	Amplify the OsMPK8 target site in this research
OsMPK8-R1	CAACCACCCATGACCATGAG	Amplify the OsMPK8 target site in this research
OsWRKY45-F1	TGCAGCCGCTCAAGGTGGAG	Amplify the OsWRKY45 target site in this research
OsWRKY45-R1	TCAAACCCATAATGTCGTCC	Amplify the OsWRKY45 target site in this research
OsCPK6-F1	AGAAACAGCATCTCTTGAGG	Amplify the OsCPK6 target site in this research
OsCPK6-R1	GCTACTTGTTGCTGTGCTTG	Amplify the OsCPK6 target site in this research
OsCPK4-F1	TTCTCATCCCACACTGCGAC	Amplify two OsCPK4 target sites in this research
OsCPK4-R1	CCGAAATGGAACTCCCGAAT	Amplify two OsCPK4 target sites in this research
OsCPK7-F1	CCTGGGAAGGATACAATTGT	Amplify the OsCPK7 target site in this research
OsCPK7-R1	TAGCTATCCCCCTGAACGTA	Amplify the OsCPK7 target site in this research
OsGSK3-F1	TGTTCTGCCTTGCCTCACTGG	Amplify the OsGSK3 target site in this research
OsGSK3-R1	ACACTGCAGAAGTTTACCTG	Amplify the OsGSK3 target site in this research
OsGSK4-F1	GGACAAAGAATGTCGCATGA	Amplify the OsGSK4 target site in this research
OsGSK4-R1	ACATGCACAAGACTAATACG	Amplify the OsGSK4 target site in this research
gOsMPK8-F1	tgttgCGGAGGAAGGTGAGAGGAGG	Target the OsMPK8（20-nt）target site
gOsMPK8-R1	aaacCCTCCTCTCACCTTCCTCCGc	Target the OsMPK8（20-nt）target site
gOsMPK8-F2	tgttgCAGACGGAGCAGCAGCAGCAGCAG	Target the OsMPK8（24-nt）target site
gOsMPK8-R2	aaacCTGCTGCTGCTGCTGCTCCGTCTGc	Target the OsMPK8（24-nt）target site
gOsWRKY45-F1	tgttgTGGAGCTACGACGCCGTCGC	Target the OsWRKY45（20-nt）target site
gOsWRKY45-R1	aaacGCGACGGCGTCGTAGCTCCAc	Target the OsWRKY45（20-nt）target site
gOsWRKY45-F2	tgttgGCACTGGAGCTACGACGCCGTCGC	Target the OsWRKY45（24-nt）target site
gOsWRKY45-R2	aaacGCGACGGCGTCGTAGCTCCAGTGCc	Target the OsWRKY45（24-nt）target site
gOsCPK6-F1	gtgtgATGGGCAACTACTACTCGTG	Target the OsCPK6 target site
gOsCPK6-R1	aaacCACGAGTAGTAGTTGCCCATc	Target the OsCPK6 target site
gOsCPK4-F1	tgttGCGGGAGCGGGAAGCGGCAG	Target the OsCPK4 T1 target site
gOsCPK4-R1	aaacCTGCCGCTTCCCGCTCCCGC	Target the OsCPK4 T1 target site
gOsCPK4-F2	gtgtgTCGCCGTCAAGCGCATCGAC	Target the OsCPK4 T2 target site
gOsCPK4-R2	aaacGTCGATGCGCTTGACGGCGAc	Target the OsCPK4 T2 target site
gOsCPK7-F1	tgttgTGCCAGAACGGGACTCTTGG	Target the OsCPK7 target site
gOsCPK7-R1	aaacCCAAGAGTCCCGTTCTGGCAc	Target the OsCPK7 target site
gOsGSK3-F1	gtgtGGAAGAATGGAGAACCTAAA	Target the OsGSK3 target site
gOsGSK3-R1	aaacTTTAGGTTCTCCATTCTTCC	Target the OsGSK3 target site
gOsGSK4-F1	tgttgAACCTGGAATACAACACCAA	Target the OsGSK4 target site
gOsGSK4-R1	aaacTTGGTGTTGTATTCCAGGTTc	Target the OsGSK4 target site
g4-F1	TAAGCTTGATATCGAATTCG	Amplify the PENTR4:gRNA4 vector to construct the PENTR4:gRNA34 plasmid
g4-R1	TAGCCAACACAAGCGGCAGC
g34-F1	CGCTGCCGCTTGTGTTGGCTAGGATCCATCGCAGTCAGCG	Amplify the sgRNA fragments to construct the PENTR4:gRNA34 plasmid
g34-R1	CGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAG

基因Gene	基因登录号Gene identifier	间隔区Spacer	PAM序列PAM sequence
OsWRKY45	LOC_Os05g25770	TGGAGCTACGACGCCGTCGC	CGG
OsWRKY45	LOC_Os05g25770	TGCACTGGAGCTACGACGCCGTCGC	CGG
OsCPK4 T1	LOC_Os02g03410	GCGGGAGCGGGAAGCGGCAG	CAG
OsCPK4 T2	LOC_Os02g03410	TCGCCGTCAAGCGCATCGAC	AAG
OsCPK6	LOC_Os02g58520	ATGGGCAACTACTACTCGTG	CGG
OsCPK7	LOC_Os03g03660	TGCCAGAACGGGACTCTTGG	GAG
OsMPK8	LOC_Os03g59390	CGGAGGAAGGTGAGAGGAGG	TGG
OsMPK8	LOC_Os03g59390	CAGACGGAGCAGCAGCAGCAGCAG	CGG
OsGSK3	LOC_Os02g14130	GGAAGAATGGAGAACCTAAA	AGG
OsGSK4	LOC_Os06g35530	AACCTGGAATACAACACCAA	AGG

基因Gene	基因登录号Gene identifier	间隔区Spacer	PAM序列PAM sequence
OsWRKY45	LOC_Os05g25770	TGGAGCTACGACGCCGTCGC	CGG
OsWRKY45	LOC_Os05g25770	TGCACTGGAGCTACGACGCCGTCGC	CGG
OsCPK4 T1	LOC_Os02g03410	GCGGGAGCGGGAAGCGGCAG	CAG
OsCPK4 T2	LOC_Os02g03410	TCGCCGTCAAGCGCATCGAC	AAG
OsCPK6	LOC_Os02g58520	ATGGGCAACTACTACTCGTG	CGG
OsCPK7	LOC_Os03g03660	TGCCAGAACGGGACTCTTGG	GAG
OsMPK8	LOC_Os03g59390	CGGAGGAAGGTGAGAGGAGG	TGG
OsMPK8	LOC_Os03g59390	CAGACGGAGCAGCAGCAGCAGCAG	CGG
OsGSK3	LOC_Os02g14130	GGAAGAATGGAGAACCTAAA	AGG
OsGSK4	LOC_Os06g35530	AACCTGGAATACAACACCAA	AGG

PAM序列 PAM Seq	基因 Gene	打靶位点 Target site	Spacer长度 Spacer lengths/bp	突变效率 Mutation efficiency	单等位突变 Mono-allelic mutation	双等位突变 Bi-allelic mutation
NGG	OsMPK8	CGGAGGAAGGTGAGAGGAGGTGG	20	5/48（10.42%）	5	0
	OsMPK8	CAGACGGAGCAGCAGCAGCAGCAGCGG	24	1/48（2.08%）	1	0
	OsWRKY45	TGGAGCTACGACGCCGTCGCCGG	20	12/48（25.00%）	11	1
	OsWRKY45	TGCACTGGAGCTACGACGCCGTCGCCGG	24	2/48（4.17%）	2	0

基于CRISPR/LanCas9的水稻基因编辑系统的开发和优化

Development and Optimization of Genome Editing in Rice with CRISPR/LanCas9 System

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PAM序列PAM Seq	基因Gene	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
NGG	OsWRKY45	25/31（80.65%）	2	23
NGG	OsCPK6	48/48（100.00%）	5	43
NAG	OsCPK4 T1	19/48（39.58%）	18	1
NAG	OsCPK7	0/48（0.00%）	0	0

PAM序列PAM Seq	基因Gene	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
NGG	OsGSK3	21/27（77.78%）	20	1
NGG	OsGSK4	26/27（96.30%）	10	16
NAG	OsCPK4 T1	14/48（29.17%）	14	0
NAG	OsCPK4 T2	0/48（0.00%）	0	0

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