通过优化gRNA设计提高CRISPR/Cas9系统中基因编辑效率的方法
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摘要
CRISPR/Cas9系统是一种广泛应用于动物、植物、真菌、细菌的新一代基因定点编辑技术,如何提高g RNA的编辑效率是该技术的关键点也是许多研究者关注的焦点。本研究利用CRISPR/Cas9技术对番茄miR167a前体序列进行基因编辑,根据番茄miR167a前体序列在线设计候选g RNA,同时结合体外编辑检测的方法筛选出能够在体外成功编辑的g RNA-G1,把其连接到含有荧光标记的pP1C.1C表达载体,转化番茄子叶,产生愈伤组织数116个,得到5个含有表达载体的材料,经测序发现,其中3个材料发生了编辑。结果表明,通过体外检测和在线软件设计相结合的方法来快速检测g RNA的编辑效率,能筛选出最优的g RNA用于载体的构建,与未采用体外编辑检测来设计g RNA的方法相比,显著提高了CRISPR/Cas9的编辑效率。本研究通过优化g RNA,创新了一种高效利用CRISPR/Cas9进行基因编辑的方法。
CRISPR/Cas9 system is a new generation approach of gene editing technology widely used in animals,plants, fungus and bacteria. However how to improve the gene editing efficiency of g RNA is the key point of this technology. In this study, the CRISPR/Cas9 technology was used to edit the tomato miR167 a precursor sequence,firstly the candidate g RNAs was designed online according to the tomato miR167 a precursor sequence, then combined with in vitro editing and detection methods to screen out the appropriate g RNA-G1, which could be successfully edited in vitro. The g RNA-G1 was ligated to pP1 C.1 C expression vector contained the fluorescent marker, and the tomato cotyledons were transformed to produce 116 callus tissues, and five materials containing the expression vector were obtained. After sequencing three materials were successfully edited. The results show that the combination of in vitro detection and online software design can quickly improve the editing efficiency of g RNA, and the optimal g RNA can be selected out for vector construction, this method significantly improved the editing efficiency of CRISPR/Cas9 compared with the method of designing g RNA without in vitro editing. This study reported an innovated method for efficient gene editing using CRISPR/Cas9 by optimized g RNA.
CRISPR/Cas9 system is a new generation approach of gene editing technology widely used in animals,plants, fungus and bacteria. However how to improve the gene editing efficiency of g RNA is the key point of this technology. In this study, the CRISPR/Cas9 technology was used to edit the tomato miR167 a precursor sequence,firstly the candidate g RNAs was designed online according to the tomato miR167 a precursor sequence, then combined with in vitro editing and detection methods to screen out the appropriate g RNA-G1, which could be successfully edited in vitro. The g RNA-G1 was ligated to pP1 C.1 C expression vector contained the fluorescent marker, and the tomato cotyledons were transformed to produce 116 callus tissues, and five materials containing the expression vector were obtained. After sequencing three materials were successfully edited. The results show that the combination of in vitro detection and online software design can quickly improve the editing efficiency of g RNA, and the optimal g RNA can be selected out for vector construction, this method significantly improved the editing efficiency of CRISPR/Cas9 compared with the method of designing g RNA without in vitro editing. This study reported an innovated method for efficient gene editing using CRISPR/Cas9 by optimized g RNA.
引文
Chen Y.C.,Wang Z.P.,Ni H.W.,Xu Y.,Chen Q.J.,and Jiang L.J.,2017,CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis,Science China Life Sciences,60(5):520-523
Douris V.,Papapostolou K.M.,Ilias A.,Roditakis E.,Kounadi S.,Riga M.,Nauen R.,and Vontas J.,2017,Investigation of the contribution of Ry R target-site mutations in diamide resistance by CRISPR/Cas9 genome modification in Drosophila,Insect.Biochem.Mol.Biol.,(87):127-135
Farboud B.,and Meyer B.J.,2015,Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design,Genetics,199(4):959-971
Heigwer F.,Kerr G.,and Boutros M.,2014,E-CRISP:fast CRISPRtarget site identification,Nature Methods,11(2):122-124
Horvath P.,and Barrangou R.,2010,CRISPR/Cas,the immune system of bacteria and archaea,Science,327(5962):167-170
Hsu P.D.,Scott D.A.,Weinstein J.A.,Ran F.A.,Konermann S.,Agarwala V.,Li Y.,Fine E.J.,Wu X.,Shalem O.,Cradick T.J.,Marraffini L.A.,Bao G.,and Zhang F.,2013,DNA targeting specificity of RNA-guided Cas9 nucleases,Nature Biotechnology,31(9):827-832
Kleinstiver B.P.,2016,High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects,Nature,529(7587):490-495
Lei Y.,Lu L.,Liu H.Y.,Li S.,Xiang F.,and Chen L.L.,2014,CRISPR-P:web tool for synthetic single-guide RNA design of CRISPR-system in plants,Mol.Plant,7(9):1494-1496
Ma Y.W.,Zhang X.,Shen B.,Lu Y.D.,Chen W.,Ma J.,Bai L.,Huang X.X.,and Zhang L.F.,2014,Generating rats with conditional alleles using CRISPR/Cas9,Cell Res.,24:122-125
Mao Y.,Zhang H.,Xu N.,Zhang B.,Guo F.,and Zhu J.,2013,Application of the CRISPR-Cas system for efficient genome engineering in plants,Molecular Plant,6(6):2008-2011
Pu Y.,Liu C.,Li J.Y.,Aerzugulitashi,Hu Y.,and Liu X.D.,2018,Different SlU6 promoters cloning and establishment of CRISPR/Cas9 mediated gene editing system in tomato,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),51(2):315-326(蒲艳,刘超,李继洋,阿尔祖古丽·塔什,胡燕,刘晓东,2018,番茄U6启动子的克隆及CRISPR/Cas9基因编辑体系的建立,中国农业科学,51(2):315-326)
Qu X.X.,2010,Genetic transformation and genetic transformation of tomato lipoxygenase gene TomloxD in Pichia Pastoris,Thesis for M.S.,Chongqing University,Supervisor:Chen G.P.,pp.25-26(屈霄霄,2010,番茄脂肪氧化酶基因TomloxD的毕赤酵母表达遗传转化及遗传转化研究,硕士学位论文,重庆大学,导师:陈国平,pp.25-26)
Sander J.D.,Maeder M.L.,Reyon D.,Voytas D.F.,Joung J.K.,and Dobbs D.,2010,Zi Fi T(Zinc Finger Targeter):an updated zinc finger engineering tool,Nucleic Acids Research,38:462-468
Wang Y.N.,Li K.X.,Chen L.,Zou Y.M.,Liu H.P.,Tian Y.P.,Li D.X.,Wang R.,Zhao F.,Ferguson B.J.,Gresshoff P.M.,and Li X.,2015,MicroRNA167-directed regulation of the auxin response factors GmARF8a and GmARF8b is required for soybean nodulation and lateral root development,Plant Physiol.,168(3):101-116
Wu M.F.,Tian Q.,and Reed J.W.,2006,Arabidopsis microR-NA167 controls patterns of ARF6 and ARF8 expression,and regulates both female and male reproduction,Development,133(21):4211-4218
Xiao Z.X.,Bai G.Z.,Zhang L.,and Li Z.J.,2009,DNA extraction of Populus euphratica based on the modified methods of CTAB,Talimu Daxue Xuebao(Journal of Tarim University),21(2):29-32(肖智样,白冠章,张玲,李志军,2009,改进的CTAB法提取胡杨(Populus euphratica)总DNA,塔里木大学学报,21(2):29-32)
Yan F.,and Zhou H.B.,2016,Overviews and applications of the CRISPR/Cas9 system in plant functional genomics and creation of new plant germplasm,Zhongguo Kexue:Shengming Kexue(Scientia Sinica Vitae),46(5):498-513(严芳,周焕斌,2016,CRISPR/Cas9技术在植物基因功能研究和新种质创制中的应用与展望,中国科学:生命科学,46(5):498-513)
Zhang J.G.,Zeng R.,Chen J.S.,Liu X.,and Liao Q.S.,2008,I-dentification of conserved microRNAs and their targets from Solanum lycopersicum Mill,Gene,423(1):1-7
Zhang Y.,Bai Y.,Wu G.,Zou S.,Chen Y.,Gao C.,and Tang D.,2017,Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat,Plant J.,91(4):714-724
Douris V.,Papapostolou K.M.,Ilias A.,Roditakis E.,Kounadi S.,Riga M.,Nauen R.,and Vontas J.,2017,Investigation of the contribution of Ry R target-site mutations in diamide resistance by CRISPR/Cas9 genome modification in Drosophila,Insect.Biochem.Mol.Biol.,(87):127-135
Farboud B.,and Meyer B.J.,2015,Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design,Genetics,199(4):959-971
Heigwer F.,Kerr G.,and Boutros M.,2014,E-CRISP:fast CRISPRtarget site identification,Nature Methods,11(2):122-124
Horvath P.,and Barrangou R.,2010,CRISPR/Cas,the immune system of bacteria and archaea,Science,327(5962):167-170
Hsu P.D.,Scott D.A.,Weinstein J.A.,Ran F.A.,Konermann S.,Agarwala V.,Li Y.,Fine E.J.,Wu X.,Shalem O.,Cradick T.J.,Marraffini L.A.,Bao G.,and Zhang F.,2013,DNA targeting specificity of RNA-guided Cas9 nucleases,Nature Biotechnology,31(9):827-832
Kleinstiver B.P.,2016,High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects,Nature,529(7587):490-495
Lei Y.,Lu L.,Liu H.Y.,Li S.,Xiang F.,and Chen L.L.,2014,CRISPR-P:web tool for synthetic single-guide RNA design of CRISPR-system in plants,Mol.Plant,7(9):1494-1496
Ma Y.W.,Zhang X.,Shen B.,Lu Y.D.,Chen W.,Ma J.,Bai L.,Huang X.X.,and Zhang L.F.,2014,Generating rats with conditional alleles using CRISPR/Cas9,Cell Res.,24:122-125
Mao Y.,Zhang H.,Xu N.,Zhang B.,Guo F.,and Zhu J.,2013,Application of the CRISPR-Cas system for efficient genome engineering in plants,Molecular Plant,6(6):2008-2011
Pu Y.,Liu C.,Li J.Y.,Aerzugulitashi,Hu Y.,and Liu X.D.,2018,Different SlU6 promoters cloning and establishment of CRISPR/Cas9 mediated gene editing system in tomato,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),51(2):315-326(蒲艳,刘超,李继洋,阿尔祖古丽·塔什,胡燕,刘晓东,2018,番茄U6启动子的克隆及CRISPR/Cas9基因编辑体系的建立,中国农业科学,51(2):315-326)
Qu X.X.,2010,Genetic transformation and genetic transformation of tomato lipoxygenase gene TomloxD in Pichia Pastoris,Thesis for M.S.,Chongqing University,Supervisor:Chen G.P.,pp.25-26(屈霄霄,2010,番茄脂肪氧化酶基因TomloxD的毕赤酵母表达遗传转化及遗传转化研究,硕士学位论文,重庆大学,导师:陈国平,pp.25-26)
Sander J.D.,Maeder M.L.,Reyon D.,Voytas D.F.,Joung J.K.,and Dobbs D.,2010,Zi Fi T(Zinc Finger Targeter):an updated zinc finger engineering tool,Nucleic Acids Research,38:462-468
Wang Y.N.,Li K.X.,Chen L.,Zou Y.M.,Liu H.P.,Tian Y.P.,Li D.X.,Wang R.,Zhao F.,Ferguson B.J.,Gresshoff P.M.,and Li X.,2015,MicroRNA167-directed regulation of the auxin response factors GmARF8a and GmARF8b is required for soybean nodulation and lateral root development,Plant Physiol.,168(3):101-116
Wu M.F.,Tian Q.,and Reed J.W.,2006,Arabidopsis microR-NA167 controls patterns of ARF6 and ARF8 expression,and regulates both female and male reproduction,Development,133(21):4211-4218
Xiao Z.X.,Bai G.Z.,Zhang L.,and Li Z.J.,2009,DNA extraction of Populus euphratica based on the modified methods of CTAB,Talimu Daxue Xuebao(Journal of Tarim University),21(2):29-32(肖智样,白冠章,张玲,李志军,2009,改进的CTAB法提取胡杨(Populus euphratica)总DNA,塔里木大学学报,21(2):29-32)
Yan F.,and Zhou H.B.,2016,Overviews and applications of the CRISPR/Cas9 system in plant functional genomics and creation of new plant germplasm,Zhongguo Kexue:Shengming Kexue(Scientia Sinica Vitae),46(5):498-513(严芳,周焕斌,2016,CRISPR/Cas9技术在植物基因功能研究和新种质创制中的应用与展望,中国科学:生命科学,46(5):498-513)
Zhang J.G.,Zeng R.,Chen J.S.,Liu X.,and Liao Q.S.,2008,I-dentification of conserved microRNAs and their targets from Solanum lycopersicum Mill,Gene,423(1):1-7
Zhang Y.,Bai Y.,Wu G.,Zou S.,Chen Y.,Gao C.,and Tang D.,2017,Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat,Plant J.,91(4):714-724