CRISPR/Cas9介导柑橘CsLOB1基因启动子的多位点编辑
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摘要
为了获得CsLOB1启动子较大片段删除的突变体,利用CRISPR/Cas9技术对CsLOB1启动子进行多位点编辑。通过在CsLOB1启动子的EBEPthA4区域及其上、下游设计不同的靶标位点,构建了2个植物表达载体pCas9CsLOB1:2sites和pCas9CsLOB1:3sites,分别对CsLOB1启动子同时进行2个位点和3个位点的编辑。测序结果表明pCas9CsLOB1:2sites和pCas9CsLOB1:3sites的基因编辑效率分别为64.7%和80.0%,突变体植株在2个sgRNA之间发生了DNA片段的删除。进一步的分析发现,不同的sgRNA具有不同的突变效率,其差异是由于不同的sgRNA对CsLOB1-识别和结合能力的差异造成的。本结果表明对CsLOB1启动子进行多位点编辑可以获得删除较大DNA片段的突变体。
To obtain mutants with large fragment deletions in the CsLOB1 promoter,in this study,we edited multiple sites of the CsLOB1 promoter using the CRISPR/Cas9 system. Two plant expression vectors,pCas9CsLOB1:2 sites and pCas9CsLOB1:3 sites,which were designed for the simultaneous targeting of two and three sites in the CsLOB1 promoter,respectively,were constructed to edit the EBEPthA4 region. Sequencing data revealed that the editing efficiency in pCas9 CsLOB1:2 sites and pCas9CsLOB1:3 sites transgenic lines was 64.7% and 80.0%,respectively,and fragment deletions between two sgRNA target sites occurred in the transgenic citrus plants. Further analyses indicated the mutation efficiency differed among the sgRNAs. The differences were likely due to variability in the binding ability of sgRNAs to the targeted CsLOB1-sequence. These results showed that mutants with the deletion of large DNA fragment can be obtained through editing multiple sites within the CsLOB1 promoter.
To obtain mutants with large fragment deletions in the CsLOB1 promoter,in this study,we edited multiple sites of the CsLOB1 promoter using the CRISPR/Cas9 system. Two plant expression vectors,pCas9CsLOB1:2 sites and pCas9CsLOB1:3 sites,which were designed for the simultaneous targeting of two and three sites in the CsLOB1 promoter,respectively,were constructed to edit the EBEPthA4 region. Sequencing data revealed that the editing efficiency in pCas9 CsLOB1:2 sites and pCas9CsLOB1:3 sites transgenic lines was 64.7% and 80.0%,respectively,and fragment deletions between two sgRNA target sites occurred in the transgenic citrus plants. Further analyses indicated the mutation efficiency differed among the sgRNAs. The differences were likely due to variability in the binding ability of sgRNAs to the targeted CsLOB1-sequence. These results showed that mutants with the deletion of large DNA fragment can be obtained through editing multiple sites within the CsLOB1 promoter.
引文
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Fan D,Liu T,Li C,Jiao Bo,Li S,Hou Y,Luo K.2015.Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation.Scientific Reports,5:12217.
Hu Y,Zhang J L,Jia H G,Sosso D,Li T,Frommer W B,Yang B,White F F,Wang N,Jones J B.2014.Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease.Proceedings of the National Academy of Sciences of the United States of America,111:E521-E529.
Jia H,Orbovic V,Jones J B,Wang N.2016.Modification of the PthA4 effector binding elements in typeⅠCsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating XccΔpthA4::dCsLOB1.3 infection.Plant Biotechnol Jorunal,14:1291-1301.
Jia H,Xu J,Orbovic V,Zhang Y,Wang N.2017.Editing citrus genome via SaCas9/sgRNA system.Frontiers in Plant Science,8:2135.
Jiang W,Zhou H,Bi H,Fromm M,Yang B,Weeks D P.2013.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice.Nucleic Acids Research,41:e188.
Li J,Norville JE,Aach J,McCormack M,Zhang D.2013.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.Nature Biotechnology,31:688-691.
Li Z,Zou L,Ye G,Xiong L,Ji Z,Zakria M,Hong N,Wang G,Chen G.2014.A potential disease susceptibility gene CsLOB of citrus is targeted by a major virulence effector PthA of Xanthomonas citri subsp.citri.Molecular Plant,7:912-915.
Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y,Xie Y,Shen R,Chen S,Wang Z,Chen Y,Guo J,Chen L,Zhao X,Dong Z,Liu Y G.2015.A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants.Molcular Plant,8:1274-1284.
Mao Y,Zhang H,Xu N,Zhang B,Gou F,Zhu J.2013.Application of the CRISPR-Cas system for efficient genome engineering in plants.Molecular Plant,6:2008-2011.
Miao J,Guo D,Zhang J,Huang Q,Qin G,Zhang X,Wan J,Gu H,Qu L J.2013.Targeted mutagenesis in rice using CRISPR-Cas system.Cell Research,23:1233-1236.
Peng A,Chen S,Lei T,Xu L,He Y,Wu L,Yao L,Zou X.2017.Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus.Plant Biotechnology Journal,15:1509-1519.
Peng A,Xu L,He Y,Lei T,Yao L,Chen S,Zou X.2015.Efficient production of marker-free transgenic‘Tarocco’blood orange(Citrus sinensis Osbeck)with enhanced resistance to citrus canker using a Cre/loxP site-recombination system.Plant Cell Tissue and Organ Culture,123:1-13.
Pennisi E.2013.The CRISPR craze.Science,341:833-836.
Shan Q,Wang Y,Li J,Zhang Y,Chen K,Liang Z,Zhang K,Liu J,Xi J J,Qiu J L,Gao C.2013.Targeted genome modification of crop plants using a CRISPR-Cas system.Nature Biotechnology,31:686-688.
Srivastava V,Underwood J L,Zhao S.2017.Dual-targeting by CRISPR/Cas9 for precise excision of transgenes from rice genome.Plant Cell Tissue and Organ Culture,129:153-160.
Wang Yuqing,Zhang Yaxian,Gao Zhipeng,Yang Wencai.2018.Breeding for resistance to tomato bacterial diseases in China:challenges and prospects.Horticultural Plant Journal,4(5):193-207.
Zhang H,Zhang J,Wei P,Zhang B,Gou Feng,Feng Z,Mao Y,Yang L,Zhang H,Xu N,Zhu J.2014.The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation.Plant Biotechnology Journal,12:797-807.
Engler C,Gruetzner R,Kandzia R,Marillonnet S.2009.Golden gate shuffling:a one-pot DNA shuffling method based on typeⅡs restriction enzymes.PLoS ONE,4(5):e5553.
Fan D,Liu T,Li C,Jiao Bo,Li S,Hou Y,Luo K.2015.Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation.Scientific Reports,5:12217.
Hu Y,Zhang J L,Jia H G,Sosso D,Li T,Frommer W B,Yang B,White F F,Wang N,Jones J B.2014.Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease.Proceedings of the National Academy of Sciences of the United States of America,111:E521-E529.
Jia H,Orbovic V,Jones J B,Wang N.2016.Modification of the PthA4 effector binding elements in typeⅠCsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating XccΔpthA4::dCsLOB1.3 infection.Plant Biotechnol Jorunal,14:1291-1301.
Jia H,Xu J,Orbovic V,Zhang Y,Wang N.2017.Editing citrus genome via SaCas9/sgRNA system.Frontiers in Plant Science,8:2135.
Jiang W,Zhou H,Bi H,Fromm M,Yang B,Weeks D P.2013.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice.Nucleic Acids Research,41:e188.
Li J,Norville JE,Aach J,McCormack M,Zhang D.2013.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.Nature Biotechnology,31:688-691.
Li Z,Zou L,Ye G,Xiong L,Ji Z,Zakria M,Hong N,Wang G,Chen G.2014.A potential disease susceptibility gene CsLOB of citrus is targeted by a major virulence effector PthA of Xanthomonas citri subsp.citri.Molecular Plant,7:912-915.
Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y,Xie Y,Shen R,Chen S,Wang Z,Chen Y,Guo J,Chen L,Zhao X,Dong Z,Liu Y G.2015.A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants.Molcular Plant,8:1274-1284.
Mao Y,Zhang H,Xu N,Zhang B,Gou F,Zhu J.2013.Application of the CRISPR-Cas system for efficient genome engineering in plants.Molecular Plant,6:2008-2011.
Miao J,Guo D,Zhang J,Huang Q,Qin G,Zhang X,Wan J,Gu H,Qu L J.2013.Targeted mutagenesis in rice using CRISPR-Cas system.Cell Research,23:1233-1236.
Peng A,Chen S,Lei T,Xu L,He Y,Wu L,Yao L,Zou X.2017.Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus.Plant Biotechnology Journal,15:1509-1519.
Peng A,Xu L,He Y,Lei T,Yao L,Chen S,Zou X.2015.Efficient production of marker-free transgenic‘Tarocco’blood orange(Citrus sinensis Osbeck)with enhanced resistance to citrus canker using a Cre/loxP site-recombination system.Plant Cell Tissue and Organ Culture,123:1-13.
Pennisi E.2013.The CRISPR craze.Science,341:833-836.
Shan Q,Wang Y,Li J,Zhang Y,Chen K,Liang Z,Zhang K,Liu J,Xi J J,Qiu J L,Gao C.2013.Targeted genome modification of crop plants using a CRISPR-Cas system.Nature Biotechnology,31:686-688.
Srivastava V,Underwood J L,Zhao S.2017.Dual-targeting by CRISPR/Cas9 for precise excision of transgenes from rice genome.Plant Cell Tissue and Organ Culture,129:153-160.
Wang Yuqing,Zhang Yaxian,Gao Zhipeng,Yang Wencai.2018.Breeding for resistance to tomato bacterial diseases in China:challenges and prospects.Horticultural Plant Journal,4(5):193-207.
Zhang H,Zhang J,Wei P,Zhang B,Gou Feng,Feng Z,Mao Y,Yang L,Zhang H,Xu N,Zhu J.2014.The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation.Plant Biotechnology Journal,12:797-807.