CN107722125A - 一种高效的人工转录激活因子dCas9‑TV及其编码基因与应用 - Google Patents
一种高效的人工转录激活因子dCas9‑TV及其编码基因与应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明涉及一种高效的人工转录激活因子dCas9‑TV及其编码基因与应用。本发明在核酸酶失活的Cas9蛋白(dCas9)的羧基端连上多个拷贝的VP64和TAL转录激活结构域，产生一系列新型的人工转录激活因子，并筛选出转录激活活性最好的dCas9‑TV。当仅使用一个向导RNA(gRNA)靶向特定基因启动子时，dCas9‑TV能高效地激活拟南芥和水稻内源基因的转录。使用多个gRNA分别靶向多个靶基因时，dCas9‑TV能同时实现多基因的转录激活。另外，dCas9‑TV被证实在人类细胞中同样具有高效的靶向转录激活活性。利用体外组装的dCas9‑TV/gRNA核糖核蛋白复合体也能对拟南芥和水稻内源基因进行转录激活。综合以上优良活性，可将其应用基因组遗传筛选、代谢产物的生物合成通路改造、作物改良等方面。
Description
技术领域
本发明涉及一种高效的人工转录激活因子dCas9-TV及其编码基因与应用，属于生物技术领域，特别是涉及基于基因过表达的农作物遗传改良和真核细胞代谢通路的人工调控。
背景技术
转录调控是真核生物基因表达的重要环节，能通过改变转录速率从而改变基因表达的水平。这一过程涉及众多转录调控元件的共同参与。其中，转录激活因子能促进RNA聚合酶和启动子的相互作用，提高靶基因的转录水平。转录激活因子含有DNA结合域(DNA-binding domain)和转录激活域(transcription activation domain，TAD)，前者决定转录激活因子结合DNA序列的特异性，后者则具有转录激活的活性。通过基因工程手段，人为地将多种转录激活域和可编程的DNA结合域重新融合，可以设计并产生出新型的人工转录激活因子。通过DNA结合域与特定的靶基因启动子DNA序列的结合，人工转录激活因子可以特异地激活特定基因的转录。近年来，由ZFN、TALEN和CRISPR/Cas9等基因编辑技术衍生的人工转录激活因子被陆续报道。这些人工转录激活因子为基因功能的研究、农作物改良、真核细胞代谢物的生产、合成生物学等领域提供了重要的技术手段。其中，CRISPR/Cas9衍生的人工转录激活因子由于其简便性和易操作性，具有最广泛的应用前景。
CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9)系统是原核生物用于抵御噬菌体或外来质粒入侵的适应性免疫系统。近年CRISPR/Cas9系统被改造成一个革命性的基因组编辑工具，包括Cas9核酸酶和向导RNA(gRNA)两个组成部分。Cas9核酸酶能与gRNA结合，gRNA通过碱基互补配对把Cas9引导至特定的DNA序列，随后Cas9发挥其核酸酶活性，使目标DNA发生双链断裂。DNA双链断裂能引发细胞的自我修复机制，如非同源末端连接(nonhomologous end joiningrepair，NHEJ)和同源重组修复(homology-directed repair，HDR)途径。前者导致双链断裂位点附近的碱基发生随机缺失或插入；后者则利用外界提供的同源DNA片段引发同源重组，产生精确的基因片段替换。两种途径都可能产生DNA序列的改变，从而达到基因编辑的目的。
Cas9蛋白中的两个核酸酶活性中心HNH和RuvC发生点突变(D10A、H840A)后，可成为失去了DNA切割能力的dCas9(dead Cas9)。dCas9仍然保持了在gRNA的引导下特异结合靶序列的能力。当把特定的转录激活结构域(如VP64、p65)与dCas9融合后，可获得新的人工转录激活因子。当其在gRNA的引导下结合到靶基因的启动子区域时，能特异地激活靶基因的转录。
目前，尽管在人和动物细胞中已先后报道SunTag、SAM、VPR等多种高效的CRISPR/Cas9衍生的人工转录激活系统，但在植物细胞中高效的人工转录激活因子仍未见报道。目前植物细胞中常用的CRISPR/Cas9转录激活因子(如dCas9-VP64)效率极低，尤其是对于细胞内源基因。当只用单个gRNA结合到靶基因启动子时，大多数情况下dCas9-VP64转录激活因子最多只能上调靶基因转录不足2倍，无法满足靶基因转录激活的需要，大大限制了该技术的应用。只有同时使用多个gRNA定位到同一个基因的启动子时，dCas9-VP64转录激活因子才能达到一定的转录激活效果。但这一方面增加了脱靶的可能性，同时又增加了实验操作的复杂性，并降低了整个转录激活系统的通量(即同时激活多个基因的能力)。
综上所述，在植物中目前仍缺乏高效的人工转录激活因子，难以实现有效的单个或多个内源基因的转录激活。同时，普遍适用于真核细胞(动植物)的人工转录激活系统也亟待开发。
发明内容
为了突破现有的技术障碍，本发明的目的是提供一种CRISPR/Cas9衍生的人工转录激活因子，通过增强其自身的转录激活活性，开发出仅使用单个gRNA便能显著激活靶基因转录的dCas9转录激活因子。本发明将大大简化单基因的靶向转录激活，从而更容易实现多基因转录激活，在基因组遗传筛选、代谢产物的生物合成通路改造、作物改良等方面具有广阔的应用前景。
本发明的另一目的是提供一种CRISPR/Cas9衍生的人工转录激活因子的应用。
本发明所采用的技术方案如下：
本发明涉及一种高效的人工转录激活因子dCas9-TV，包含一个核酸酶失活的Cas9蛋白结构域和一个复合型转录激活结构域；该复合型转录激活结构域包含多个拷贝的VP64和TAL转录激活序列。
进一步地，所述的人工转录激活因子dCas9-TV，该蛋白质的氨基酸序列如序列表SEQ ID NO.1所示。
进一步地，所述的人工转录激活因子dCas9-TV，其编码基因的核苷酸序列如序列表SEQ ID NO.2所示。
本发明还提供一种基于dCas9-TV的人工基因转录激活系统，该系统包括上述人工转录激活因子dCas9-TV和由RNA聚合酶III型启动子驱动表达的gRNA；gRNA特异性靶向靶基因的启动子，数目可以为一个或多个。
进一步地，所述的人工基因转录激活系统，该系统可在拟南芥和水稻等植物细胞以及人类细胞中应用，特异激活靶基因的表达。
本发明还提供一种体外组装的dCas9-TV/gRNA核糖核蛋白转录激活因子复合体，该复合体包括重组表达并纯化的人工转录激活因子dCas9-TV，其氨基酸序列如序列表SEQID NO.1所示；同时该系统还包括体外转录纯化的gRNA，gRNA特异性靶向靶基因的启动子。
本发明还提供一个可用于特异性激活水稻内源GW7基因转录的gRNA，该gRNA对应的DNA序列如序列表SEQ ID NO.3所示。
本发明还提供一个可用于特异性激活水稻内源ER1基因转录的gRNA，该gRNA对应的DNA序列如序列表SEQ ID NO.4所示。
与现有技术相比，本发明具有以下有益效果：
1)本发明的CRISPR/Cas9衍生的人工转录激活因子dCas9-TV能在仅使用一个gRNA的情况下高效地激活植物和人类细胞内源基因的转录，同时也能使用多个gRNA分别靶向多个启动子以同时实现多基因的转录激活，而体外组装的dCas9-TV/gRNA核糖核蛋白复合体也可成功地激活细胞内源基因的转录，同时排除分子克隆的需要，有效降低技术门槛。
2)本发明的新型人工转录激活因子dCas9-TV较目前常用的dCas9-VP64在转录激活效率上有了很大提高，在基础研究、药物筛选和生产、农作物改良等方面有着广泛的应用前景。
附图说明
图1为CRISPR/Cas9衍生的人工转录激活因子结构示意图；dCas9为核酸酶失活的Cas9，NLS为核定位信号，VP64和TAL是两种转录激活结构域。FLAG蛋白标签便于dCas9-TV蛋白的western blot检测。
图2为4种dCas9人工转录激活因子在拟南芥原生质体中对WRKY30-LUC萤光素酶报告基因的转录激活效果比较以及SDS-PAGE电泳结果；Ctrl为空白对照。
图3为5种dCas9人工转录激活因子在拟南芥原生质体中对WRKY30-LUC萤光素酶报告基因的转录激活效果比较以及SDS-PAGE电泳结果；Ctrl为空白对照。
图4为使用不同的拟南芥U6启动子表达gRNA对dCas9-TV转录激活效果的影响；Ctrl为空白对照。
图5为dCas9-TV在拟南芥转基因植株中对内源WRKY30基因的转录激活。
图6为dCas9-TV在拟南芥原生质体中对RLP23-LUC报告基因的激活；Ctrl为空白对照。
图7为dCas9-TV在拟南芥原生质体中对内源RLP23基因的转录激活；Ctrl为空白对照。
图8为dCas9-TV在拟南芥转基因植株中对内源RLP23基因的转录激活。
图9为dCas9-TV/gRNA-RLP23转基因植株受nlp20诱导后MAPK的激活情况；RuBisCo为上样量对照。
图10为dCas9-TV/gRNA-RLP23转基因植株受nlp20诱导后产生的活性氧爆发。
图11为dCas9-TV在拟南芥原生质体中的多基因转录激活；Ctrl为空白对照。
图12为dCas9-TV在水稻原生质体中的多基因转录激活；Ctrl为空白对照。
图13为dCas9-TV在人类细胞中激活内源基因的转录。
图14a为dCas9-TV-His重组蛋白的表达和纯化电泳图，CBB staining代表考马斯亮蓝染色，Protein marker代表蛋白分子量标准，*所示条带为目的蛋白。
图14b为体外转录的gRNA的电泳结果，Ethedium bromide staining代表溴化乙锭染色，RNA marker代表单链RNA分子量标准，箭头所示条带为目的条带。
图15为基于体外组装的dCas9-TV/gRNA核糖核蛋白复合体的转录激活。dCas9-TVonly代表只转染dCas9-TV重组蛋白，RNP代表转染dCas9-TV/gRNA核糖核蛋白复合体。
具体实施方式
本发明涉及一个高效的人工转录激活因子dCas9-TV，以及该转录激活因子在动植物细胞中的应用。
本发明对Cas9蛋白进行定点突变，获得核酸酶失活的dCas9，并在其羧基端连接多个VP64和TAL转录激活结构域，在拟南芥原生质体系统中，利用LUC报告系统筛选出高效的人工转录激活因子dCas9-TV，其转录激活活性较dCas9-VP64有了很大提高。
在只使用一个gRNA时，dCas9-TV能在拟南芥原生质体以及转基因植株中显著地上调内源WRKY30基因的转录水平，与目前常用的dCas9-VP64相比转录激活效率有了很大的提高。另外，本发明还选择拟南芥RLP23基因验证dCas9-TV的性能，在原生质体和转基因植株中，RLP23基因的转录被dCas9-TV显著激活；同时，还对上述转基因植株进行表型分析，发现RLP23的转录水平上升后，植株对免疫信号nlp20的免疫应答明显增强。
本发明还表明在使用多个gRNA分别靶向多个基因启动子时，dCas9-TV能同时激活拟南芥或水稻多个内源基因的转录，而dCas9-VP64则无法实现多基因的转录激活。
除了植物外，dCas9-TV还能在人类细胞中高效地激活靶基因的转录。
本发明还提供一种体外组装的dCas9-TV/gRNA核糖核蛋白转录激活因子复合体，使用重组表达的dCas9-TV和体外转录所得的gRNA组装的核糖核蛋白复合体，能在植物细胞中成功地激活靶基因的转录。
以下通过具体较佳实施例结合附图进一步说明本发明，但本发明并不仅限于以下的实施例。
实施例1
CRISPR/Cas9衍生的人工转录激活因子的构建
通过PCR定点突变技术将pcoCas9(plant codon-optimized SpCas9)的第10位氨基酸“D”和第840位氨基酸“H”的密码子先后替换为氨基酸“A”的密码子，获得核酸酶失活的Cas9，即dCas9。所使用的引物如下所示：
Cas9-D10A-F：GTACTCTATCGGACTTGCTATCGGAACCAACTCTG
Cas9-D10A-R：CAGAGTTGGTTCCGATAGCAAGTCCGATAGAGTAC
Cas9-H840A-F：TGATTACGATGTTGATGCTATCGTTCCACAGTCTT
Cas9-H840A-R：AAGACTGTGGAACGATAGCATCAACATCGTAATCA
Q5高保真PCR聚合酶购自NEB公司，定点突变PCR反应体系如下：
HBT-pcoCas9质粒由本实验室构建和保存。反应程序：98℃变性30s；98℃变性10s，55℃退火30s，72℃延伸10min，扩增16个循环；最后72℃延伸10min。向所得PCR产物中加入DpnI(购自NEB公司)0.5μl消化过夜，产物转化大肠杆菌感受态细胞，挑取单克隆，经氨苄青霉素抗性筛选和sanger测序验证定点突变，得到HBT-dCas9。
利用含有VP64转录激活域编码序列的长引物，以HBT-dCas9为模板进行PCR反应，得到dCas9-VP64编码片段，该片段经BamHI和PstI(NBE公司)酶切后用T4DNA连接酶(NEB公司)连接到同样经BamHI和PstI消化的HBT载体上，连接产物转化大肠杆菌并经氨苄青霉素抗性筛选阳性克隆子，最终通过测序验证得到dCas9-VP64表达载体HBT-dCas9-VP64。其中，dCas9与VP64中间引入了AvrII的酶切位点。所用PCR引物如下：
dCas9-BamHI-F：CGAGGATCCATGGATTACAAGGA
dCas9-VP64-PstI-R：CGACTGCAGTCAAAGCATATCCAAGTCGAAATCATCAAGGGCGTCAGATCCAAGCATATCCAAGTCGAAATCATCAAGGGCGTCAGATCCAAGCATATCCAAGTCGAAATCATCAAGGGCGTCAGATCCAAGCATATCCAAGTCGAAATCATCAAGGGCGTCAGATCCCCTAGGCTTCTTCTTCTTAGCCTGTCC
在HBT-dCas9-VP64的基础上构建dCas9-VP128、dCas9-VP192和dCas9-VP256的表达载体。VP64的编码序列由美国IDT公司化学合成，合成片段的5’端和3’端分别带有AvrII和NheI酶切位点。用AvrII和NheI(NEB公司)双酶切VP64合成片段产生粘性末端，并与经AvrII线性化的HBT-dCas9-VP64进行T4连接反应。连接产物转化大肠杆菌，氨苄青霉素筛选后对阳性克隆提取质粒进行酶切鉴定，最终测序验证，得到HBT-dCas9-VP128。其中，VP64片段5’端的AvrII与载体的AvrII连接后保留了AvrII酶切位点，而3’端的NheI与载体的AvrII连接后则破坏了这两种酶切位点，即最终在dCas9和VP128中间保留有一个AvrII酶切位点。进而，利用该AvrII酶切位点，在HBT-dCas9-VP128基础上再插入一个和两个经AvrII和NheI消化的VP64合成片段，则可得到HBT-dCas9-VP192和HBT-dCas9-VP256。
进而，在HBT-dCas9-VP128的基础上进一步构建dCas9-2TAL-VP128、dCas9-4TAL-VP128、dCas9-6TAL-VP128(即dCas9-TV)、dCas9-8TAL-VP128的表达载体。其中“TAL”表示来源于黄单胞菌的转录激活域。构建方式类似于上述在HBT-dCas9-VP64的基础上构建dCas9-VP128、dCas9-VP192和dCas9-VP256的方法。具体地，2TAL(2个TAL串联而成)的编码序列由美国IDT化学合成，合成片段的5’端和3’端都分别带有AvrII和NheI酶切位点。HBT-dCas9-VP128载体用AvrII线性化处理后，在dCas9和VP128之间先后插入1个、2个、3个和4个经AvrII、NheI酶切的2TAL编码片段，得到HBT-dCas9-2TAL-VP128、HBT-dCas9-4TAL-VP128、HBT-dCas9-TV、HBT-dCas9-8TAL-VP128。其中，重组质粒HBT-dCas9-TV包含如SEQ ID NO.2所示的基因。
为了在人类细胞中表达上述人工转录激活因子，VP64和TV的编码序列经PCR扩增后均在5’端和3’端分别带上AvrII和XbaI酶切位点，随后PCR产物经AvrII和XbaI酶切后与经XbaI线性化处理的pcDNA3.1-dCas9(Addgene plasmid#47106)载体连接，得到pcDNA3.1-dCas9-VP64和pcDNA3.1-dCas9-TV。其中，pcDNA3.1-dCas9-TV包含如SEQ IDNO.2所示的基因。
所构建的CRISPR/Cas9衍生的人工转录激活因子结构示意图如图1所示，dCas9为核酸酶失活的Cas9，NLS为核定位信号，VP64和TAL是两种转录激活结构域。
实施例2
基于人工转录激活因子dCas9-TV的拟南芥WRKY30基因的转录激活
(1)拟南芥WRKY30基因启动子靶序列的设计及gRNA表达载体的构建
从NCBI数据库查找拟南芥WRKY30基因的启动子序列，并在转录起始位点上游200bp内挑选靶序列“AAGAACGAAGAAAGCTGATGTGG”(下划线为PAM结构)，并据此设计gRNA-WRKY30。对于此基因，本发明使用两种表达框AtU6-1:gRNA:TTTTTT和AtU6-26:gRNA:TTTTTT，其构建均使用重叠PCR方法，重叠序列为20nt靶序列。AtU6-1和AtU6-26启动子先前已由本实验室克隆所得。构建AtU6-1:gRNA:TTTTTT所用PCR引物如下：
U6-1-SacI-F：CGAGAGCTCAGAAATCTCAAAATTCCG
U6-1-WRKY30-R：CATCAGCTTTCTTCGTTCTTCAATCACTACTTCGTCTCT
gRNA-WRKY30-F：AAGAACGAAGAAAGCTGATGGTTTTAGAGCTAGAAATAGC
gRNA-SacI-R：CGAGAGCTCAAAAAAGCACCGACTCGGTGC
构建AtU6-26:gRNA:TTTTTT所用PCR引物如下：
U6-26-SacI-F：CGAGAGCTCAGCTTTTTTTCTTCTTCT
U6-26-WRKY30-R：CATCAGCTTTCTTCGTTCTTCAATCACTACTTCGACTCT
gRNA-WRKY30-F：AAGAACGAAGAAAGCTGATGGTTTTAGAGCTAGAAATAGC
gRNA-SacI-R：CGAGAGCTCAAAAAAGCACCGACTCGGTGC
重叠PCR终产物经跑胶分离后，用OMEGA公司的Cycle Pure Kit按使用说明纯化。纯化产物用SacI(NEB公司)酶切后，与经SacI线性化的pUC119-RCS进行连接反应。连接产物转化大肠杆菌，用氨苄青霉素筛选阳性克隆子，提取质粒进行酶切鉴定，最终通过测序验证gRNA表达载体的成功构建，得到AtU6-1:gRNA-WRKY30和AtU6-26:gRNA-WRKY30质粒。
(2)拟南芥原生质体的制备和转染
拟南芥原生质体的制备方法如下。
取健康的、长势良好的4周龄土培拟南芥(Col-0)植株的叶片，置于实验台上的干净A4纸上，用锋利的刀片把切成约1mm宽的长条，并迅速放入含有10ml酶解液(1.5％纤维素酶R10，0.4％果胶酶R10，0.4M甘露醇，20mM MES(pH 5.7)，20mM KCl，10mM CaCl2和0.1％BSA)的培养皿中，使叶条完全浸没在其中，室温避光消化3h。随后把培养皿置于水平摇床上以60rpm转速摇荡3min。加入10ml W5溶液(154mM NaCl，125mM CaCl2，5mM KCl和2mM MES(pH 5.7))并轻轻摇匀，吸取酶切产物并用洁净的尼龙网滤去未被消化的叶片组织，滤液收集于30ml圆底离心管中，置于水平离心机中100g离心2min，吸走上清。再次加入10ml W5溶液，重新悬浮，冰上静置30min。随后在水平离心机中100g离心2min，吸去上清，加入适量MMG溶液(0.4M甘露醇，15mM MgCl2和4mM MES(pH5.7))重悬原生质体，在普通光学显微镜下用血球计数板确定细胞密度，最终把细胞密度调整至每毫升2×105个原生质体，即可进行转染。
拟南芥原生质体的转染方法如下。
在2ml圆底离心管中，加入200μl拟南芥原生质体与8μl(16μg)dCas9-TAD表达载体或空白载体、8μl(16μg)gRNA-WRKY30表达载体、4μl(8μg)WRKY30-LUC质粒和1μl(2μg)UBQ10-GUS质粒，再加入220μl PEG溶液(40％PEG4000(v/v)，0.2M甘露醇和0.1M CaCl2)，轻柔地充分混匀，室温静置转染5min，随后加入800μl W5溶液，混匀以终止转染。水平离心机100g离心2min，吸去上清，加入100μl W5溶液重悬，并转移至1ml WI溶液(0.5M甘露醇，20mMKCl和4mM MES(pH 5.7))，室温黑暗培养12h。
(3)萤光素酶报告基因活性的检测
转染后的拟南芥原生质体黑暗培养12h后，100g水平离心2min，吸去上清，加入100μl裂解液(25mM Tris-HCl(pH 7.8)，2mM DTT，2mM trans-1,2-diaminocyclohexane-N’N’N’N’-tetraacetic acid，10％(v/v)glycerol和1％(v/v)Triton X-100)，剧烈震荡使原生质体充分裂解。
取10μl裂解产物于96孔板中，加入100μl虫萤光素溶液(20mM Tricine，1.07mM(MgCO3)4Mg(OH)2，2.67mM MgSO4，0.1mM EDTA，33.3mM DTT，270μM辅酶A，0.47mMD-虫萤光素钠盐，0.53mM ATP，pH 7.8)，迅速置于酶标仪中读取时长为1秒的萤光强度，即LUC读数。同时，另取10μl裂解产物于96孔板中，加入50μl MUG溶液(10mM Tris-HCl(pH 8.0)，1mM 4-methylumbelliferyl-β-D-glucuronide(MUG)，2mM MgCl2)，37℃敷育30min后，置于冰水浴中冷却以终止反应，随后放入酶标仪中读取萤光强度，即GUS读数。各个样品报告基因的活性表示为LUC读数与GUS读数的比值LUC/GUS。最后剩余裂解产物用于进行SDS-PAGE和Western blot检测各个人工转录激活因子的表达情况。
实验结果见图2、图3和图4。图2结果表明当使用AtU6-1:gRNA-WRKY30靶向WRKY30-LUC时，与空白对照相比，dCas9-VP64、dCas9-VP128和dCas9-VP192均能上调报告基因的表达水平，其中dCas9-VP128的转录激活效果最佳(5.1倍)，优于目前植物细胞中常用的dCas9-VP64。dCas9-VP192和dCas9-VP256效果欠佳的可能原因是其重复序列过多而导致的蛋白稳定性较差或表达量较低。图3则进一步测试了以dCas9-VP128为基础而构建的一系列人工转录激活因子的活性，结果表明TAL转录激活域的增加能进一步增强转录激活活性。这些转录激活因子均能显著地激活报告基因的表达水平，其中dCas9-6TAL-VP128(即dCas9-TV)的活性最好(55.6倍)，因此本发明后续主要阐述dCas9-TV的使用。图4显示与AtU6-1启动子相比，使用AtU6-26启动子表达gRNA时能大大提升dCas9-TV/gRNA系统在拟南芥中的转录激活活性。
(4)转基因拟南芥植株的获得
首先构建用于遗传转化拟南芥植株的双元载体。以HBT-dCas9-TV和HBT-dCas9-VP64表达载体作为模板，通过PCR扩增35SPPDK:dCas9-TV:NOS和35SPPDK:dCas9-VP64:NOS表达框，所用引物如下：
HBT-35SPPDK-StuI-F：CGAAGGCCTTACTCCAAGAATATCAAAGAT
HBT-NOS-StuI-R：CGAAGGCCTGATCTAGTAACATAGATGACA
扩增产物经StuI(NEB公司)酶切后，连接至双元载体pFGC-RCS的StuI位点。连接产物转化大肠杆菌感受态细胞，经卡那霉素筛选和测序验证后得到pFGC-dCas9-TV和pFGC-dCas9-VP64质粒。随后AtU6-26:gRNA-WRKY30表达框用AscI(NEB公司)从pUC119载体上酶切出来，并分别与经AscI酶线性化的pFGC-dCas9-TV和pFGC-dCas9-VP64载体进行连接反应。连接产物转化大肠杆菌感受态细胞，经卡那霉素筛选和测序验证后最终得到用于遗传转化的pFGC-dCas9-TV-gRNA-WRKY30和pFGC-dCas9-VP64-gRNA-WRKY30质粒。
将上述最终双元载体通过电击转化农杆菌(Agrobacterium tumefaciens)株系GV3101中，利用花粉管导入法(floral dip)转化拟南芥植株。具体地，将含有目标双元载体的GV3101菌液，按1:100的比例接种于含卡那霉素(50mg/L)的液体LB培养基中，在28℃摇床中以转速220rpm培养2天。5000g收集菌体，弃去培养基，加入含0.05％Silwet L77的5％蔗糖溶液重悬。取已开花的拟南芥植株，倒置并使花序完全浸入农杆菌菌液中，轻轻搅动约10s后取出，置于湿润黑暗环境中1天后，转移至正常生长环境，直到收获成熟种子。种子播种于含有1:1000稀释的Basta除草剂(Amersco公司)的土壤中，筛选具有Basta抗性的转基因植株。
(5)转基因拟南芥植株WRKY30基因转录水平的检测
取6周龄具Basta除草剂抗性的转基因植株或野生型植株，剪取约20mg叶片，用RNAisoPlus试剂(TaKaRa公司)按产品说明书提取叶片总RNA。取1μg总RNA经RNase-freeDNase I(NEB公司)处理去除基因组DNA后，使用Transcriptor First Strand cDNASynthesis Kit(Roche公司)根据产品说明书进行逆转录反应。所得cDNA稀释后用FastStart Essential DNA Green Master试剂(Roche公司)在LightCycler 96Instrument(Roche公司)中进行RT-qPCR反应。以拟南芥ACT2基因作为内参基因，运用2-ΔΔCt法计算WRKY30基因的转录激活水平。所用的qPCR引物如下：
WRKY30-qPCR-F：TCGGAGCCAAATTTCCAAGAGG
WRKY30-qPCR-R：TCCTCGGTAACTGATCTCAAGGAG
ACT2-qPCR-F：GACCTTTAACTCTCCCGCTATG
ACT2-qPCR-R：AAACCCTCGTAGATTGGCAC
实验结果(图5)显示，与野生型植株相比，dCas9-VP64仅能微弱(#13)或不能(#15)激活拟南芥内源WRKY30基因的转录。相反，dCas9-TV在转基因拟南芥植株中具有优良的转录激活活性，能显著把内源WRKY30基因的转录水平上调至47.5-138.8倍。可见本发明公开的人工转录激活因子dCas9-TV比目前常用的dCas9-VP64具有更强的转录激活活性。
实施例3
基于人工转录激活因子dCas9-TV的拟南芥RLP23基因的转录激活
(1)拟南芥RLP23基因启动子靶序列的设计及gRNA表达载体的构建
从NCBI数据库查找拟南芥RLP23基因的启动子序列，并在转录起始位点上游200bp内挑选靶序列“AAATCCCTTAACGTATAACTTGG”(下划线为PAM结构)，并据此设计gRNA-RLP23。使用重叠PCR方法构建AtU6-26:gRNA:TTTTTT表达框，重叠序列为20nt靶序列。所用PCR引物如下：
U6-26-SacI-F：CGAGAGCTCAGCTTTTTTTCTTCTTCT
U6-26-RLP23-R：AGTTATACGTTAAGGGATTTCAATCACTACTTCGACTCT
gRNA-RLP23-F：AAATCCCTTAACGTATAACTGTTTTAGAGCTAGAAATA
gRNA-SacI-R：CGAGAGCTCAAAAAAGCACCGACTCGGTGC
重叠PCR终产物经跑胶分离后，用OMEGA公司的Cycle Pure Kit按使用说明纯化。纯化产物用SacI(NEB公司)酶切后，与经SacI线性化的pUC119-RCS进行连接反应。连接产物转化大肠杆菌，用氨苄青霉素筛选阳性克隆子，提取质粒进行酶切鉴定，最终通过测序验证gRNA表达载体的成功构建，得到AtU6-26:gRNA-RLP23质粒。
(2)拟南芥原生质体的制备和转染
拟南芥原生质体的制备同实施例2中(2)，方法不变。
转染原生质体时，测量萤光素酶报告基因活性的转染方法与实施例2中(2)相同，将其中的基因WRKY30改成RLP23。测量细胞内源RLP23基因转录激活水平的转染方法与实施例2中(2)相同，但转染体系变为：400μl原生质体、20μl(40μg)dCas9-TAD表达载体和20μl(40μg)gRNA表达载体。其他溶液的用量加倍。
(3)萤光素酶报告基因活性的检测
方法同实施例2中(3)。实验结果见图6，表明在单一一种靶定RLP23启动子的gRNA的引导下，dCas9-TV能显著激活RLP23-LUC报告基因的表达，达到28.5倍；而dCas9-VP64仅能微弱地上调报告基因的表达(1.4倍)，活性欠佳。
(4)原生质体内源RLP23基因转录激活水平的检测
400μl原生质体黑暗培养12h后，收集于离心管中，100g水平离心2min，吸去上清。加入1ml RNAiso Plus试剂按产品说明书提取总RNA。取1μg总RNA经RNase-free DNase I处理去除基因组DNA后，使用Transcriptor First Strand cDNA Synthesis Kit根据产品说明书进行逆转录反应。所得cDNA稀释后用FastStart Essential DNA Green Master试剂在LightCycler96Instrument中进行RT-qPCR反应。以拟南芥ACT2基因作为内参基因，运用2-ΔΔCt法计算目的基因的转录激活水平。所用的qPCR引物如下：
RLP23-qPCR-F：GCTCTGTGATGGTGCCTCTT
RLP23-qPCR-R：AAGCTTTGAGCCAGAACGGA
ACT2-qPCR-F：GACCTTTAACTCTCCCGCTATG
ACT2-qPCR-R：AAACCCTCGTAGATTGGCAC
图7中实验结果显示，在转染后的原生质体中，dCas9-TV能高效地激活拟南芥内源RLP23基因的转录至44.2倍。相反，dCas9-VP64非但不能有效增强转录，反而微弱地降低了RLP23的转录水平(0.7倍)。此结果再次证明dCas9-TV是比dCas9-VP64更高效的CRISPR/Cas9衍生的人工转录激活因子。
(5)转基因拟南芥植株的获得
方法同实施例2中(4)，将其中的基因WRKY30全部改成RLP23。
(6)转基因拟南芥植株RLP23基因转录水平的检测
方法同实施例2中(5)，将其中的基因WRKY30全部改成RLP23，相应地，使用到的RT-qPCR引物改成实施例3中(4)所用引物。
实验结果如图8所示。图8显示dCas9-VP64在三个不同株系中均不能引起内源RLP23基因的转录激活，甚至产生了转录抑制(#1和#5)。而dCas9-TV在四个不同株系中均能使内源RLP23基因的转录水平提高超过30倍，再次说明dCas9-TV在转基因植株中具有优良的转录激活活性。综合以上结果，新型人工转录激活因子dCas9-TV能在细胞和转基因植株中显著地激活单个靶基因的转录水平，转录激活性能远好于目前广泛使用的dCas9-VP64。
(7)转基因拟南芥植株表型分析
本实施例中的靶基因RLP23编码的蛋白质是一个模式识别受体(patternrecognition receptor，PRR)，能识别微生物相关分子模式(microbe-associatedmolecular pattern，MAMP)nlp20。nlp20是含20个氨基酸的肽段，是一种来源于细菌、真菌和卵菌的保守分子元件。当病原菌入侵拟南芥植株时，位于拟南芥细胞膜上的RLP23蛋白能感知和识别病原菌的nlp20分子模式，并启动下游免疫信号转导，从而引起模式触发式免疫(Pattern-triggered Immunity，PTI)应答，以抵御入侵病原菌。因此推测植物细胞RLP23蛋白的增多将使细胞感知和识别免疫信号nlp20的能力增强，所引起的免疫应答也随之增强，最终获得更强的抗病性。测试
实施例3(6)中转基因拟南芥植株对nlp20免疫信号的响应情况，包括MAPK激活和活性氧(ROS)的产生。
取6周龄野生型和转基因拟南芥植株(dCas9-TV/gRNA-RLP23#1、#9、#10、#14)，按实施例2中(2)分别制备原生质体。6×104个(300μl)原生质体经100g水平离心2min后，吸走上清，加入100μl W5溶液重悬，并转移至1ml WI溶液中，室温培养3h。加入10nM化学合成的nlp20肽段，对照组加入等体积灭菌水，处理10min后离心收集样品。向样品中加入SDS上样缓冲液并混匀，置于95℃加热3min，随后样品用于进行10％SDS-PAGE和Western blot，用anti-phospho ERK antibodies(Cell Signaling Technology公司)作为一抗、HRP-conjugated anti-rabbit antibodies(Cell Signaling Technology公司)作为二抗检测MAPK信号。
实验结果如图9所示，低浓度的nlp20信号仅能微弱地激活野生型植株的MAPK信号，而转基因植株(dCas9-TV/gRNA-RLP23#1、#9、#10、#14)的MAPK信号则被明显地激活。该结果表明，dCas9-TV能通过提高转基因植株内源RLP23基因的转录水平(图8)，使转基因植株对低浓度的免疫信号变得更加敏感，增强了植株的免疫响应。
取6周龄野生型和转基因拟南芥植株(dCas9-TV/gRNA-RLP23#1、#9、#10、#14)，用打孔器取下直径5mm的叶圆片，每个植株各取下4个。向96孔板的各孔中加入200μl无菌水，把每个叶圆片分别放置其中，室温静置过夜。吸走无菌水，加入100μl含有1μM nlp20肽段的HRP-luminol反应液，立即置于酶标仪中测量萤光强度。每孔每次读取1s内的累积萤光信号，每隔30s读取一次，共读取100次。数据整理后得到活性氧产生的时间序列图如图10，可见转基因植株(dCas9-TV/gRNA-RLP23#1、#9、#10、#14)感知免疫信号nlp20后产生了比野生型植株更多的活性氧，预示着转基因植株对相应病原菌具有更强的防御反应。
实施例4
基于人工转录激活因子dCas9-TV的拟南芥基因WRKY30、RLP23和CDG1的多基因转
录激活
(1)靶基因启动子靶序列的设计及gRNA表达载体的构建
拟南芥WRKY30基因启动子靶序列的设计及gRNA表达载体的构建同实施例2中(1)。
拟南芥RLP23基因启动子靶序列的设计及gRNA表达载体的构建同实施例3中(1)。
拟南芥CDG1基因启动子靶序列的设计及gRNA表达载体的构建同实施例3中(1)，所使用的体系、方法均不变，将其中的基因RLP23全部改成CDG1。相应地，把靶序列“AAATCCCTTAACGTATAACTTGG”改为靶序列“TTGTAGATTATTACCCTCAATGG”；所用引物“U6-26-RLP23-R”改为“U6-26-CDG1-R”，引物“gRNA-RLP23-F”改为“gRNA-CDG1-F”，引物序列具体如下：
U6-26-CDG1-R：TTGAGGGTAATAATCTACAACAATCACTACTTCGACTCT
gRNA-CDG1-F：TTGTAGATTATTACCCTCAAGTTTTAGAGCTAGAAATA
最终得到AtU6-26:gRNA-CDG1质粒。
(2)拟南芥原生质体的制备和转染
拟南芥原生质体的制备同实施例2中(2)，方法不变。
拟南芥原生质体的转染方法与实施例2中(2)相同，但转染体系变为:400μl原生质体、20μl(40μg)dCas9-TAD表达载体、7μl(14μg)gRNA-WRKY30表达载体、7μl(14μg)gRNA-RLP23表达载体和7μl(14μg)gRNA-CDG1表达载体。其他溶液的用量加倍。
(3)原生质体靶基因转录激活水平的检测
原生质体靶基因转录水平的检测方法同实施例3中(4)。所用RT-qPCR引物如下：
WRKY30-qPCR-F：TCGGAGCCAAATTTCCAAGAGG
WRKY30-qPCR-R：TCCTCGGTAACTGATCTCAAGGAG
RLP23-qPCR-F：GCTCTGTGATGGTGCCTCTT
RLP23-qPCR-R：AAGCTTTGAGCCAGAACGGA
CDG1-qPCR-F：GGAGCTTATCAGTGGACGCA
CDG1-qPCR-R：AACAACGGTCGTGCCCAAT
ACT2-qPCR-F：GACCTTTAACTCTCCCGCTATG
ACT2-qPCR-R：AAACCCTCGTAGATTGGCAC
实验结果见图11，使用3个gRNA分别靶向3个基因的启动子时，dCas9-VP64只能微弱地上调拟南芥内源CDG1基因的转录水平(3.2倍)，而对另外两个基因没有转录激活效果。相反，dCas9-TV能同时显著地激活上述3个基因的转录，表明新型人工转录激活因子dCas9-TV在拟南芥细胞中能实现高效的多基因转录激活。
实施例5
基于人工转录激活因子dCas9-TV的水稻基因GW7和ER1的多基因转录激活
(1)靶基因启动子靶序列的设计及gRNA表达载体的构建
水稻GW7基因的靶序列设计及gRNA表达载体的构建如下。从NCBI数据库查找水稻GW7基因的启动子序列，并在转录起始位点上游200bp内挑选靶序列“TGTCCAGCTCCCGGCACCCACGG”(下划线为PAM结构)，并据此设计gRNA-GW7。使用重叠PCR方法构建OsU6a:gRNA:TTTTTT表达框，重叠序列为20nt靶序列。OsU6a启动子获取于华南农业大学刘耀光实验室。所用PCR引物如下：
OsU6a-KpnI-F：CGAGGTACCTTTTTTCCTGTAGTTTTCCCACAAC
OsU6a-GW7-R：TGGGTGCCGGGAGCTGGACACGGCAGCCAAGCCAGCACCC
gRNA-GW7-F：TGTCCAGCTCCCGGCACCCAGTTTTAGAGCTAGAAATAGC
gRNA-HindIII-R：CGAAAGCTTAAAAAAGCACCGACTCGGTGCC
重叠PCR终产物经跑胶分离后，用OMEGA公司的Cycle Pure Kit按使用说明纯化。纯化产物用KpnI和HindIIII(NEB公司)酶切后，与经KpnI和HindIIII线性化的pUC119-RCS进行连接反应。连接产物转化大肠杆菌，用氨苄青霉素筛选阳性克隆子，提取质粒进行酶切鉴定，最终通过测序验证gRNA表达载体的成功构建，得到OsU6a:gRNA-GW7质粒，该质粒包含如SEQ ID NO.3所示的基因。
水稻ER1基因的靶序列设计及gRNA表达载体的构建方法同上，把基因GW7全部改成ER1。相应地，把靶序列“TGTCCAGCTCCCGGCACCCACGG”改为“GTTCTACTCCTACCAACTCTAGG”，启动子“OsU6a”变成“OsU6b”。OsU6b启动子获取于华南农业大学刘耀光实验室。所用引物变成如下：
OsU6b-KpnI-F：CGAGGTACCTGCAAGAACGAACTAAGCCG
OsU6b-ER1-R：AGAGTTGGTAGGAGTAGAACAACACAAGCGGCAGCGCGC
gRNA-ER1-F：GTTCTACTCCTACCAACTCTGTTTTAGAGCTAGAAATAGC
gRNA-HindIII-R：CGAAAGCTTAAAAAAGCACCGACTCGGTGCC
最终得到OsU6b:gRNA-ER1质粒，该质粒包含如SEQ ID NO.4所示的基因。
(2)水稻原生质体的制备和转染
水稻原生质体的制备方法同实施例2中(2)，把“4周龄土培拟南芥(Col-0)植株的叶片”变为“10天龄水稻(中花11号)幼苗的秸秆”、“长条”变为“薄粒”、“100g离心2min”变为“200g离心5min”。
水稻原生质体的转染方法与实施例2中(2)相似。转染体系为：400μl原生质体、20μl(40μg)dCas9-TAD表达载体、10μl(20μg)gRNA-GW7表达载体和10μl(20μg)gRNA-ER1表达载体。其他溶液的用量加倍。“室温静置5min”改为“室温避光静置15min”、100g离心2min”变为“200g离心5min”
(3)原生质体靶基因转录激活水平的检测
原生质体靶基因转录水平的检测方法同实施例3中(4)，内参基因“拟南芥ACT2”改为“水稻ACT1”。所用RT-qPCR引物如下：
OsGW7-qPCR-F：CATCGACACCAAGTCACAAGGG
OsGW7-qPCR-R：TGACGTGTCGAGGACAGAGATG
OsER1-qPCR-F：CTGTAGCCCACGGATAATACAC
OsER1-qPCR-R：GCCATAGCTGTAGACATCAGAC
OsACT1-qPCR-F:CCACTATGTTCCCTGGCATT
OsACT1-qPCR-R：GTACTCAGCCTTGGCAATCC
检测结果如图12，在水稻原生质体中dCas9-TV能同时显著地上调靶基因GW7和ER1的转录水平，而dCas9-VP64则仅能微弱地激活GW7基因的转录，对ER1基因则有转录抑制效果。上述结果表明，新型人工转录激活因子dCas9-TV在水稻中也能特异地激活靶基因的转录，并且具有优良的多基因激活的性能。
实施例6
基于人工转录激活因子dCas9-TV的人类ASCL1和OCT4基因的转录激活
(1)靶基因启动子靶序列的设计及gRNA表达载体的构建
人类ASCL1基因的靶序列设计及gRNA表达载体的构建同实施例3中(1)类似，把“拟南芥”改成“人类”，“RLP23”改成“ASCL1”。相应地，靶序列“AAATCCCTTAACGTATAACTTGG”改成“GCAGCCGCTCGCTGCAGCAGCGG”，“AtU6-26:gRNA:TTTTTT”改成“HsU6:gRNA:TTTTTTT”，所用引物改成如下：
U6-SacI-F：CGAGAGCTCGAGGGCCTATTTCCCATGAT
U6-ASCL1-R：CTGCTGCAGCGAGCGGCTGGTCCTTTCCACAAGATATATAAAGC
gRNA-ASCL1-F：GCAGCCGCTCGCTGCAGCAGGTTTTAGAGCTAGAAATAGC
gRNA-SacI-R：CGAGAGCTCAAAAAAGCACCGACTCGGTGC
最终得到HsU6:gRNA-ASCL1质粒。
人类OCT4基因的靶序列设计及gRNA表达载体的构建方法同上，把基因ASCL1全部改成OCT4。相应地，把靶序列“GCAGCCGCTCGCTGCAGCAGCGG”改为“ACACCATTGCCACCACCATTAGG”。所用引物变成如下：
U6-SacI-F：CGAGAGCTCGAGGGCCTATTTCCCATGAT
U6-OCT4-R：AATGGTGGTGGCAATGGTGTCGTCCTTTCCACAAGATATATAAAGC
gRNA-OCT4-F：GACACCATTGCCACCACCATTGTTTTAGAGCTAGAAATAGC
gRNA-SacI-R：CGAGAGCTCAAAAAAGCACCGACTCGGTGC
最终得到HsU6:gRNA-OCT4质粒。
(2)细胞培养和转染
人类HEK293T细胞在含有10％FBS和1％青霉素的DMEM培养液中传代，置于37℃和5％CO2的培养环境中。进行DNA转染前一天，1.6×105个细胞接种于24孔板中。利用Lipofectamine LTX with PLUS(Thermo Fisher公司)把500ng gRNA表达载体、500ngdCas9-TAD表达载体和30ng p-GFP质粒转染至细胞中，培养3天。
(3)靶基因转录激活水平的检测
收取HEK293T细胞，使用RNeasy Plus RNA isolation kit(Qiagen公司)按产品说明书提取总RNA，随后使用One Step SYBR PrimeScript RT-PCR kit(TaKaRa公司)按产品说明书进行一步法RT-qPCR，所用仪器为LightCycler 480Instrument(Roche公司)。以人类GAPDH基因作为内参基因，运用2-ΔΔCt法计算目的基因的转录激活水平。所用的qPCR引物如下：
HsOCT4-qPCR-F：GCTCGAGAAGGATGTGGTCC
HsOCT4-qPCR-R：CGTTGTGCATAGTCGCTGCT
HsASCL1-qPCR-F：GGAGCTTCTCGACTTCACCA
HsASCL1-qPCR-R：AACGCCACTGACAAGAAAGC
HsGAPDH-qPCR-F：CGAGATCCCTCCAAAATCAA
HsGAPDH-qPCR-R：ATCCACAGTCTTCTGGGTGG
检测结果如图13，与对照相比，dCas9-TV能分别使内源基因ASCL1和OCT4的转录水平提升至46.0倍和16.4倍，表明dCas9-TV在人类HEK293T细胞中也发挥特异性转录激活的作用。与dCas9-VP64相比，dCas9-TV的转录激活活性有了很大的提高。
实施例7
基于体外组装的dCas9-TV/gRNA核糖核蛋白转录激活因子复合体的拟南芥和水稻
基因的转录激活
(1)dCas9-TV人工转录激活因子的表达和纯化
将dCas9-TV基因克隆进专用的6×His标签原核表达载体。dCas9-TV-His表达载体转化入大肠杆菌Rosetta感受态细胞(Transgen公司)，使用2×YT培养基于37℃，220rpm培养至OD600＝0.6-0.8。加入终浓度为0.2mM的IPTG，于18℃以转速220rpm振荡培养16h。随后于4℃5000g离心10min，收集菌体并去上清，加1×PBS(140mM NaCl，3mM KCl，1.7mMNa2HPO4，pH 7.4)重悬菌体。再于4℃5000g离心10min，弃上清。向上述菌体中按照10ml/g加入His标签裂解液(300mM NaCl，50mM NaH2PO4，20mM imidazole，10mM Tris-base，pH 8.0)，于4℃重悬菌体。加入适量溶菌酶，使用超声破碎仪破碎30min至菌液大体澄清。于4℃，7000g离心30min收取上清液，带His标签的重组蛋白溶解在上清中。向前述上清液中加入Ni-NTA珠子(Transgen公司)，置于4℃60rpm振荡孵育2-3h，重组蛋白结合在珠子上。将上述孵育液缓缓倒入层析柱，液体自然流出。之后，先用His标签洗涤液(300mM NaCl，50mMNaH2PO4，50mM imidazole，10mM Tris-base，pH 8.0)洗涤3遍，每次10ml，弃流出液。再使用His标签洗脱液(300mM NaCl,50mM NaH2PO4，200mM imidazole，10mM Tris-base，10％glycerol，pH 8.0)进行重组蛋白的洗脱，目的重组蛋白在洗脱液中。最后，使用超滤管(50K，Milipore公司)对上述洗脱下的蛋白进行更换缓冲液(20mM HEPES pH 7.5，150mMKCl，1mM DTT，5％glycerol)以及浓缩，最终dCas9-TV-6×His重组蛋白浓度调至约750μg/ml。通过SDS-PAGE检测重组蛋白的表达情况(图14a)，重组蛋白大小符合预期。
(2)gRNA的体外转录和纯化
本发明以拟南芥WRKY30、RLP23基因和水稻ER1基因为例阐述体外组装的人工转录激活因子RNP复合体的性能。分别取实施例2中(1)AtU6-26:gRNA-WRKY30质粒、实施例3中(1)AtU6-26:gRNA-RLP23质粒和实施例5中(1)OsU6b:gRNA-ER1质粒作模板，利用PCR扩增出3种“T7启动子-靶序列-gRNA骨架”片段，所用引物如下：
T7p-WRKY30-F：AAGCTAATACGACTCACTATAGGAAGAACGAAGAAAGCTGATG
T7p-RLP23-F：AAGCTAATACGACTCACTATAGGAAATCCCTTAACGTATAACT
T7p-ER1-F：AAGCTAATACGACTCACTATAGGTTCTACTCCTACCAACTCT
gRNA-R：AAAAGCACCGACTCGGTGCCACTT
使用E.Z.N.A Gel Extraction Kit(OMEGA公司)纯化上述“T7启动子-靶序列-gRNA骨架”片段。取75ng纯化后的PCR产物作为模板，使用HiScribe Quick T7High YieldRNA SynthesisKit(NEB公司)按照说明书进行体外转录反应。所得产物用RNase-freeDNase I(NBE公司)除去DNA模板后，利用RNA Clean&Concentrator-25试剂盒(ZymoResearch公司)纯化gRNA。所得3种gRNA通过尿素变性PAGE确认其长度和完整性(图14b)，条带大小均符合预期，约为97bp。
(3)dCas9-TV/gRNA核糖核蛋白(RNP)复合体的体外组装
对于特定的靶基因，取RNase-free的1.5ml离心管，先后加入40μl(30μg)dCas9-TV蛋白和10μl(25μg)相应的gRNA，轻轻吹打混匀，室温静置10min即可。对照组用等体积的RNase-free水替代gRNA。组装好的dCas9-TV/gRNA RNP立即用于原生质体转染。
(4)原生质体的制备和转染
拟南芥原生质体的制备同实施例2中(2)，所用方法不变，所用试剂均用RNase-free水配制。水稻原生质体的制备同实施例5中(2)，所用方法不变，所用试剂均用RNase-free水配制。
体外组装的dCas9-TV/gRNA RNP通过PEG介导转染400μl原生质体。拟南芥原生质体的转染方法同实施例2中(2)，方法不变，体系扩大1倍。水稻原生质体的转染方法同实施例5中(2)，方法不变，体系扩大1倍。转染后的原生质体于室温暗培养5h，即可检测靶基因转录水平。
(5)靶基因转录水平的检测
拟南芥WRKY30和RLP23转录水平的检测方法同实施例3中(4)，使用实施例4(3)中对应的RT-qPCR引物。水稻ER1转录水平的检测方法同实施例5中(3)，使用实施例5中(3)对应的RT-qPCR引物。
实验结果如图15所示，证明了体外组装的dCas9-TV/gRNA RNP复合体能在拟南芥和水稻中特异地激活靶基因的转录。
本发明并不局限于上述实施方式，如果对本发明的各种改动或变型不脱离本发明的精神和范围，倘若这些改动和变型属于本发明的权利要求和等同技术范围之内，则本发明也意图包含这些改动和变动。
SEQUENCE LISTING
<110> 中山大学
<120> 一种高效的人工转录激活因子dCas9-TV及其编码基因与应用
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<170> PatentIn version 3.5
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<223> dCas9-TV（dCas9-6TAL-VP128）
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Lys Lys Lys Lys Pro Arg Gly Gly Ser Gly Gly Leu Leu Asp Pro
1415 1420 1425
Gly Thr Pro Met Asp Ala Asp Leu Val Ala Ser Ser Thr Val Val
1430 1435 1440
Trp Glu Gln Asp Ala Asp Pro Phe Ala Gly Thr Ala Asp Asp Phe
1445 1450 1455
Pro Ala Phe Asn Glu Glu Glu Leu Ala Trp Leu Met Glu Leu Leu
1460 1465 1470
Pro Gln Gly Gly Ser Gly Gly Leu Leu Asp Pro Gly Thr Pro Met
1475 1480 1485
Asp Ala Asp Leu Val Ala Ser Ser Thr Val Val Trp Glu Gln Asp
1490 1495 1500
Ala Asp Pro Phe Ala Gly Thr Ala Asp Asp Phe Pro Ala Phe Asn
1505 1510 1515
Glu Glu Glu Leu Ala Trp Leu Met Glu Leu Leu Pro Gln Ala Arg
1520 1525 1530
Gly Gly Ser Gly Gly Leu Leu Asp Pro Gly Thr Pro Met Asp Ala
1535 1540 1545
Asp Leu Val Ala Ser Ser Thr Val Val Trp Glu Gln Asp Ala Asp
1550 1555 1560
Pro Phe Ala Gly Thr Ala Asp Asp Phe Pro Ala Phe Asn Glu Glu
1565 1570 1575
Glu Leu Ala Trp Leu Met Glu Leu Leu Pro Gln Gly Gly Ser Gly
1580 1585 1590
Gly Leu Leu Asp Pro Gly Thr Pro Met Asp Ala Asp Leu Val Ala
1595 1600 1605
Ser Ser Thr Val Val Trp Glu Gln Asp Ala Asp Pro Phe Ala Gly
1610 1615 1620
Thr Ala Asp Asp Phe Pro Ala Phe Asn Glu Glu Glu Leu Ala Trp
1625 1630 1635
Leu Met Glu Leu Leu Pro Gln Ala Arg Gly Gly Ser Gly Gly Leu
1640 1645 1650
Leu Asp Pro Gly Thr Pro Met Asp Ala Asp Leu Val Ala Ser Ser
1655 1660 1665
Thr Val Val Trp Glu Gln Asp Ala Asp Pro Phe Ala Gly Thr Ala
1670 1675 1680
Asp Asp Phe Pro Ala Phe Asn Glu Glu Glu Leu Ala Trp Leu Met
1685 1690 1695
Glu Leu Leu Pro Gln Gly Gly Ser Gly Gly Leu Leu Asp Pro Gly
1700 1705 1710
Thr Pro Met Asp Ala Asp Leu Val Ala Ser Ser Thr Val Val Trp
1715 1720 1725
Glu Gln Asp Ala Asp Pro Phe Ala Gly Thr Ala Asp Asp Phe Pro
1730 1735 1740
Ala Phe Asn Glu Glu Glu Leu Ala Trp Leu Met Glu Leu Leu Pro
1745 1750 1755
Gln Ala Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ala
1760 1765 1770
Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp
1775 1780 1785
Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe
1790 1795 1800
Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu
1805 1810 1815
Asp Met Leu Ala Arg Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu
1820 1825 1830
Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met
1835 1840 1845
Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly
1850 1855 1860
Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu
1865 1870 1875
<210> 2
<211> 5628
<212> DNA
<213> Artificial
<220>
<221> CDS
<222> (1)...(5628)
<223> dCas9-TV（dCas9-6TAL-VP128）
<400> 2
atggattaca aggatgatga tgataaggat tacaaggatg atgatgataa gatggctcca 60
aagaagaaga gaaaggttgg aatccacgga gttccagctg ctgataagaa gtactctatc 120
ggacttgcta tcggaaccaa ctctgttgga tgggctgtta tcaccgatga gtacaaggtt 180
ccatctaaga agttcaaggt tcttggaaac accgatagac actctatcaa gaagaacctt 240
atcggtgctc ttcttttcga ttctggagag accgctgagg ctaccagatt gaagagaacc 300
gctagaagaa gatacaccag aagaaagaac agaatctgct accttcagga aatcttctct 360
aacgagatgg ctaaggttga tgattctttc ttccacagac ttgaggagtc tttccttgtt 420
gaggaggata agaagcacga gagacaccca atcttcggaa acatcgttga tgaggttgct 480
taccacgaga agtacccaac catctaccac cttagaaaga agttggttga ttctaccgat 540
aaggctgatc ttagacttat ctaccttgct cttgctcaca tgatcaagtt cagaggacac 600
ttccttatcg agggagacct taacccagat aactctgatg ttgataagtt gttcatccag 660
cttgttcaga cctacaacca gcttttcgag gagaacccaa tcaacgcttc tggagttgat 720
gctaaggcta tcctttctgc tagactttct aagtctcgta gacttgagaa ccttatcgct 780
cagcttccag gagagaagaa gaacggactt ttcggaaacc ttatcgctct ttctcttgga 840
cttaccccaa acttcaagtc taacttcgat cttgctgagg atgctaagtt gcagctttct 900
aaggatacct acgatgatga tcttgataac cttcttgctc agatcggaga tcagtacgct 960
gatcttttcc ttgctgctaa gaacctttct gatgctatcc ttctttctga catccttaga 1020
gttaacaccg agatcaccaa ggctccactt tctgcttcta tgatcaagag atacgatgag 1080
caccaccagg atcttaccct tttgaaggct cttgttagac agcagcttcc agagaagtac 1140
aaggaaatct tcttcgatca gtctaagaac ggatacgctg gatacatcga tggaggagct 1200
tctcaggagg agttctacaa gttcatcaag ccaatccttg agaagatgga tggaaccgag 1260
gagcttcttg ttaagttgaa cagagaggat cttcttagaa agcagagaac cttcgataac 1320
ggatctatcc cacaccagat ccaccttgga gagcttcacg ctatccttcg tagacaggag 1380
gatttctacc cattcttgaa ggataacaga gagaagatcg agaagatcct taccttcaga 1440
atcccatact acgttggacc acttgctaga ggaaactctc gtttcgcttg gatgaccaga 1500
aagtctgagg agaccatcac cccttggaac ttcgaggagg ttgttgataa gggagcttct 1560
gctcagtctt tcatcgagag aatgaccaac ttcgataaga accttccaaa cgagaaggtt 1620
cttccaaagc actctcttct ttacgagtac ttcaccgttt acaacgagct taccaaggtt 1680
aagtacgtta ccgagggaat gagaaagcca gctttccttt ctggagagca gaagaaggct 1740
atcgttgatc ttcttttcaa gaccaacaga aaggttaccg ttaagcagtt gaaggaggat 1800
tacttcaaga agatcgagtg cttcgattct gttgaaatct ctggagttga ggatagattc 1860
aacgcttctc ttggaaccta ccacgatctt ttgaagatca tcaaggataa ggatttcctt 1920
gataacgagg agaacgagga catccttgag gacatcgttc ttacccttac ccttttcgag 1980
gatagagaga tgatcgagga gagactcaag acctacgctc accttttcga tgataaggtt 2040
atgaagcagt tgaagagaag aagatacacc ggatggggta gactttctcg taagttgatc 2100
aacggaatca gagataagca gtctggaaag accatccttg atttcttgaa gtctgatgga 2160
ttcgctaaca gaaacttcat gcagcttatc cacgatgatt ctcttacctt caaggaggac 2220
atccagaagg ctcaggtttc tggacaggga gattctcttc acgagcacat cgctaacctt 2280
gctggatctc cagctatcaa gaagggaatc cttcagaccg ttaaggttgt tgatgagctt 2340
gttaaggtta tgggtagaca caagccagag aacatcgtta tcgagatggc tagagagaac 2400
cagaccaccc agaagggaca gaagaactct cgtgagagaa tgaagagaat cgaggaggga 2460
atcaaggagc ttggatctca aatcttgaag gagcacccag ttgagaacac ccagcttcag 2520
aacgagaagt tgtaccttta ctaccttcag aacggaagag atatgtacgt tgatcaggag 2580
cttgacatca acagactttc tgattacgat gttgatgcta tcgttccaca gtctttcttg 2640
aaggatgatt ctatcgataa caaggttctt acccgttctg ataagaacag aggaaagtct 2700
gataacgttc catctgagga ggttgttaag aagatgaaga actactggag acagcttctt 2760
aacgctaagt tgatcaccca gagaaagttc gataacctta ccaaggctga gagaggagga 2820
ctttctgagc ttgataaggc tggattcatc aagagacagc ttgttgagac cagacagatc 2880
accaagcacg ttgctcagat ccttgattct cgtatgaaca ccaagtacga tgagaacgat 2940
aagttgatca gagaggttaa ggttatcacc ttgaagtcta agttggtttc tgatttcaga 3000
aaggatttcc agttctacaa ggttagagag atcaacaact accaccacgc tcacgatgct 3060
taccttaacg ctgttgttgg aaccgctctt atcaagaagt acccaaagtt ggagtctgag 3120
ttcgtttacg gagattacaa ggtttacgat gttagaaaga tgatcgctaa gtctgagcag 3180
gagatcggaa aggctaccgc taagtacttc ttctactcta acatcatgaa cttcttcaag 3240
accgagatca cccttgctaa cggagagatc agaaagagac cacttatcga gaccaacgga 3300
gagaccggag agatcgtttg ggataaggga agagatttcg ctaccgttag aaaggttctt 3360
tctatgccac aggttaacat cgttaagaaa accgaggttc agaccggagg attctctaag 3420
gagtctatcc ttccaaagag aaactctgat aagttgatcg ctagaaagaa ggattgggac 3480
ccaaagaagt acggaggatt cgattctcca accgttgctt actctgttct tgttgttgct 3540
aaggttgaga agggaaagtc taagaagttg aagtctgtta aggagcttct tggaatcacc 3600
atcatggagc gttcttcttt cgagaagaac ccaatcgatt tccttgaggc taagggatac 3660
aaggaggtta agaaggatct tatcatcaag ttgccaaagt actctctttt cgagcttgag 3720
aacggaagaa agagaatgct tgcttctgct ggagagcttc agaagggaaa cgagcttgct 3780
cttccatcta agtacgttaa cttcctttac cttgcttctc actacgagaa gttgaaggga 3840
tctccagagg ataacgagca gaagcagctt ttcgttgagc agcacaagca ctaccttgat 3900
gagatcatcg agcaaatctc tgagttctct aagagagtta tccttgctga tgctaacctt 3960
gataaggttc tttctgctta caacaagcac agagataagc caatcagaga gcaggctgag 4020
aacatcatcc accttttcac ccttaccaac cttggtgctc cagctgcttt caagtacttc 4080
gataccacca tcgatagaaa aagatacacc tctaccaagg aggttcttga tgctaccctt 4140
atccaccagt ctatcaccgg actttacgag accagaatcg atctttctca gcttggagga 4200
gataagagac cagctgctac caagaaggct ggacaggcta agaagaagaa gcctagggga 4260
ggatctggtg gtcttttgga tccaggaacc cctatggatg ctgaccttgt tgcttcttcc 4320
accgttgtct gggagcagga tgccgaccct ttcgctggta ccgcagacga cttcccagct 4380
tttaacgagg aagagcttgc ttggctcatg gaattgttgc cacagggagg ttctggtgga 4440
ttgttggatc ctggaactcc aatggacgct gatcttgttg cttcctctac tgtcgtttgg 4500
gagcaagacg ctgacccttt cgctggaacc gctgacgatt tcccagcctt taacgaagag 4560
gaattggcct ggcttatgga gttgcttcct caggctaggg gaggatctgg tggtcttttg 4620
gatccaggaa cccctatgga tgctgacctt gttgcttctt ccaccgttgt ctgggagcag 4680
gatgccgacc ctttcgctgg taccgcagac gacttcccag cttttaacga ggaagagctt 4740
gcttggctca tggaattgtt gccacaggga ggttctggtg gattgttgga tcctggaact 4800
ccaatggacg ctgatcttgt tgcttcctct actgtcgttt gggagcaaga cgctgaccct 4860
ttcgctggaa ccgctgacga tttcccagcc tttaacgaag aggaattggc ctggcttatg 4920
gagttgcttc ctcaggctag gggaggatct ggtggtcttt tggatccagg aacccctatg 4980
gatgctgacc ttgttgcttc ttccaccgtt gtctgggagc aggatgccga ccctttcgct 5040
ggtaccgcag acgacttccc agcttttaac gaggaagagc ttgcttggct catggaattg 5100
ttgccacagg gaggttctgg tggattgttg gatcctggaa ctccaatgga cgctgatctt 5160
gttgcttcct ctactgtcgt ttgggagcaa gacgctgacc ctttcgctgg aaccgctgac 5220
gatttcccag cctttaacga agaggaattg gcctggctta tggagttgct tcctcaggct 5280
aggggtggat ctggaggtgg tggtagcgga ggagatgctc ttgatgattt cgatcttgat 5340
atgcttggat ctgacgctct cgacgacttt gacctcgaca tgttgggatc tgatgctctt 5400
gacgatttcg atttggacat gttgggttct gacgccttgg acgatttcga cctcgacatg 5460
cttgctaggg gatctgacgc ccttgatgat ttcgacttgg atatgcttgg atctgacgcc 5520
cttgatgatt tcgacttgga tatgcttgga tctgacgccc ttgatgattt cgacttggat 5580
atgcttggat ctgacgccct tgatgatttc gacttggata tgctttga 5628
<210> 3
<211> 96
<212> DNA
<213> Artificial
<220>
<221> misc_feature
<222> (1)...(96)
<223> gRNA-GW7
<400> 3
tgtccagctc ccggcaccca gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 96
<210> 4
<211> 96
<212> DNA
<213> Artificial
<220>
<221> misc_feature
<222> (1)...(96)
<223> gRNA-ER1
<400> 4
gttctactcc taccaactct gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 96
Claims (8)
1.一种高效的人工转录激活因子dCas9-TV，其特征在于，包含一个核酸酶失活的Cas9蛋白结构域和一个复合型转录激活结构域；该复合型转录激活结构域包含多个拷贝的VP64和TAL转录激活序列。
2.如权利要求1所述的人工转录激活因子dCas9-TV，其特征在于，该蛋白质的氨基酸序列如序列表SEQ ID NO.1所示。
3.如权利要求1所述的人工转录激活因子dCas9-TV，其特征在于，其编码基因的核苷酸序列如序列表SEQ ID NO.2所示。
4.一种基于dCas9-TV的人工基因转录激活系统，其特征在于，该系统包括如权利要求1所述的人工转录激活因子dCas9-TV和由RNA聚合酶III型启动子驱动表达的gRNA；所述gRNA特异性靶向靶基因的启动子，数目可以为一个或多个。
5.如权利要求4所述的人工基因转录激活系统，其特征在于，该系统可在拟南芥和水稻等植物细胞以及人类细胞中应用，特异激活靶基因的表达。
6.一种体外组装的dCas9-TV/gRNA核糖核蛋白转录激活因子复合体，其特征在于，该复合体包含如权利要求1所述的重组表达并纯化的人工转录激活因子dCas9-TV和体外转录并纯化的gRNA。
7.可用于特异性激活水稻内源GW7基因转录的gRNA，其特征在于，该gRNA对应的DNA序列如序列表SEQ ID NO.3所示。
8.可用于特异性激活水稻内源ER1基因转录的gRNA，其特征在于，该gRNA对应的DNA序列如序列表SEQ ID NO.4所示。
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| WO2022116139A1 (zh) * | 2020-12-04 | 2022-06-09 | 中国科学院脑科学与智能技术卓越创新中心 | 一种检测细胞内源性低丰度基因和lncRNA水平的方法 |
| CN113637658A (zh) * | 2021-08-19 | 2021-11-12 | 中国农业科学院棉花研究所 | 基于dCas9-oToV的基因转录系统及其应用 |
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| CN114958910A (zh) * | 2022-05-13 | 2022-08-30 | 南方医科大学 | DropCRISPRa高效靶向相分离基因激活系统及其构建方法和应用 |
| CN114958910B (zh) * | 2022-05-13 | 2024-03-01 | 南方医科大学 | DropCRISPRa高效靶向相分离基因激活系统及其构建方法和应用 |
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