CN104513814A - I-TevIN201-Cas9null融合蛋白及其应用 - Google Patents
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Abstract
本发明涉及一种I-TevIN201-Cas9null融合蛋白及其应用，属于分子生物学领域，本发明通过将I-TevIN201的N端201氨基酸和Cas9null（没有切割活性）的N端融合，得到一种I-TevIN201-Cas9null融合蛋白，该蛋白在gRNA的引导下，可以序列特异性的切割CNNNG位点，达到精确切割基因组的作用，从而使细胞的脱靶效应大大降低；此外利用本发明所得I-TevIN201-Cas9null融合蛋白做药物筛选或基因修饰时更安全，该蛋白还可用于基因治疗。
Description
技术领域
本发明属于分子生物学领域，具体涉及一种I-TevIN201-Cas9null融合蛋白及其应用。
背景技术
CRISPR-Cas源自细菌和古细菌的免疫系统，可利用靶点特异性的RNA将Cas9核酸酶带到基因组上的具体靶点，从而对特定基因位点进行修饰。RNA导向Cas9可以在多种细胞和生物体中发挥功能，在特异位点裂解完整基因组。Cas9这种基因组编辑的潜能使这项技术已经广泛用于人的细胞系，小鼠，大鼠，多物种的基因敲除及敲入。
I-TevI是一种归巢酶，它有N末端的催化结构域，中间的链接区域和C末端的DNA结合域构成。该酶序列特异性的识别CNNNG序列。
特异性的gRNA靶向的Cas9基因敲除技术已经有了比较广的应用，但是该技术存在很大的不足，就是脱靶效应。为了提高打靶的特异性，有很多改进，比如选择多个gRNA打靶，然后进行脱靶效应分析，此外，还有用两个突变型的Cas9即 Cas9 nickase，用两个gRNA引导Cas9 nickase去进行切割。还有的科学家用失活的Cas9即Cas9null，连接一个ForkI蛋白（Cas9null-ForkI），对特定基因位点进行修饰，但是仍然存在脱靶效应。为了最大程度上提高其特异性，我们用失活的Cas9即Cas9null和特异性识别CNNNG序列的I-TevIN201蛋白融合，形成一个gRNA靶向的，特异性切割CNNNG序列工具酶，该工具酶可以对基因组进行精确的基因编辑。
发明内容
解决的技术问题：
本发明的目的在于克服现有技术的不足提供一种I-TevIN201-Cas9null融合蛋白，即将I-TevIN201的N端201氨基酸和Cas9null（没有切割活性）的N端融合。该蛋白在gRNA的引导下，可以序列特异性的切割CNNNG位点，达到精确切割基因组的作用，使产生的脱靶效应大大降低。
技术方案：
I-TevIN201-Cas9null融合蛋白，所述融合蛋白为由SEQ ID NO.1所示氨基酸序列组成的蛋白。
所述的I-TevIN201-Cas9null融合蛋白的核苷酸序列。
以上所述的核苷酸序列如SEQ ID NO.2所示。
所述的I-TevIN201-Cas9null融合蛋白，其第十位天冬氨酸突变成丙氨酸，第839位组氨酸突变成丙氨酸，第840位组氨酸突变成丙氨酸，第863位天冬酰胺突变成丙氨酸，该突变蛋白没有核酸酶活性。
所述的I-TevIN201-Cas9null融合蛋白，其中I-TevIN201蛋白由归巢酶I-TevI N末端201个氨基酸组成，包含I-TevI的酶的催化活性区域和连接域。
所述的I-TevIN201-Cas9null融合蛋白在药物筛选及基因修饰中的应用。
所述的I-TevIN201-Cas9null融合蛋白的基因可以克隆到真核表达载体上，用于细胞和动物的基因修饰。
有益效果
本发明通过将I-TevIN201的N端201氨基酸和Cas9null（没有切割活性）的N端融合，得到一种I-TevIN201-Cas9null融合蛋白，该蛋白在gRNA的引导下，可以序列特异性的切割CNNNG位点，达到精确切割基因组的作用，从而使细胞的脱靶效应大大降低；此外利用本发明所得I-TevIN201-Cas9null融合蛋白做药物筛选或基因修饰时更安全，该蛋白还可用于基因治疗。
现有的技术对基因组切割都是序列非特异的切割，gRNA的引导下，Cas9蛋白可以在gRNA序列上，序列两边做随意的切割。而本发明I-TevIN201-Cas9null融合蛋白可以精确的进行序列特异性的切割，在gRNA的引导下，特异性的识别切割CNNNG序列。所以，它的特异性和可控性非常大，因此，本发明的应用更广，更实用。
附图说明
图1为I-TevIN201-Cas9null蛋白活性的验证图，由图可见为I-TevIN201-Cas9null蛋白与线性化的质粒pDNA3.1，tracrRNA，Mg2+，gRNA孵育这一组，DNA有切割活性，其他组没有切割活性；
图2为PCR扩增Srebf2基因，回收PCR产物的测序结果图（野生型对照）；
图3为I-TevIN201-Cas9null蛋白对Srebf2基因切割活性验证图，将I-TevIN201-Cas9null真核表达载体和gRNA载体同时转染小鼠L929细胞，经过Hygromycin筛选，挑取一个单克隆。回收PCR产物送测序，结果显示与野生型对照对比， I-TevIN201-Cas9null蛋白对Srebf2基因都有切割活性。
图4 为I-TevIN201-Cas9null蛋白对Srebf2基因切割活性验证图，将I-TevIN201-Cas9null真核表达载体和gRNA载体同时转染小鼠L929细胞，经过Hygromycin筛选，随机再挑取第二个单克隆。回收PCR产物送测序，结果显示与野生型对照对比， I-TevIN201-Cas9null蛋白对Srebf2基因都有切割活性。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。但本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明中，引物Primer_F、引物Primer_ R、引物I-TevIN201-Cas9null _F: NdeI、引物I-TevIN201-Cas9null _R：、各引物购于生工生物工程（上海）股份有限公司；原核表达载体pET-16b购于Novagen；大肠杆菌E. coli BL21购于Takara；IPTG购于Takara；胶回收试剂盒购自天根生化科技(北京)有限公司；小鼠L929细胞购于博研(上海)生物科技有限公司，I-TevIN201-Cas9nullDNA序列是委托生工生物工程(上海)股份有限公司合成的。
实施例1 I-TevI
N201
-Cas9
null
蛋白的表达与纯化
方法如下：
第一部分：I-TevIN201-Cas9null真核表达载体的构建：
序列合成I-TevIN201-Cas9null，以I-TevIN201-Cas9nullDNA序列（SEQ ID NO.2）为模板，用引物Primer_F：CAGCCTCCGGACTCTAGAATGAAAAGCGGAATTTATCAGAT（SEQ ID NO.3）和引物Primer_ R： AACTCATTACTAACCGGTCACCTTCCTCTTCTTCTTGGGGTC（SEQ ID NO.4）扩增I-TevIN201-Cas9null，克隆到hCas9载体的XbaI&AgeI位点，构建成pI-TevIN201-Cas9null载体。
具体的扩增步骤如下：
PCR扩增I-TevIN201-Cas9null，配置反应体系和反应程序如下：
表1 反应体系
| 5×primerSTAR Buffer | 10 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq HS (5 U/μl) | 0.5ul |
| Primer_F1 20pmol (final conc. 0.4 μM) | 1ul |
| Primer_ R1 20pmol (final conc. 0.4 μM) | 1ul |
| I-TevIN201-Cas9null (20ng/ul) | 1ul |
| ddH2O | 32.5 ul |
| 总计 | 50 ul |
扩增条件：98℃ 10s，61℃ 30s，72℃，4.5min，共30个循环。
胶回收I-TevIN201-Cas9null片段，用XbaI&AgeI酶切hCas9载体，胶回收骨架载体；两个片段进行In-Fusion反应（Clothech），转化，克隆鉴定，找到pI-TevIN201-Cas9null阳性克隆。
第二部分：pET-16b-I-TevIN201-Cas9null原核核表达载体的构建：
用引物I-TevIN201-Cas9null _F: NdeI：
TCGAAGGTCGTCATATGATGAAAAGCGGAATTTATCAGAT（SEQ ID NO.5）和I-TevIN201-Cas9null _R:BamHI：TTGTTAGCAGCCGGATCCTCACCTTCCTCTTCTTCTTGGGGTC（SEQ ID NO.6）去扩增I-TevIN201-Cas9null，
配置反应体系和反应程序如下：
表2 反应体系
| 5×primerSTAR Buffer | 10 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq HS (5 U/μl) | 0.5ul |
| I-TevIN201-Cas9null _F: NdeI 20pmol | 1ul |
| I-TevIN201-Cas9null _R:BamHI 20pmol | 1ul |
| I-TevIN201-Cas9null (20ng/ul) | 1ul |
| ddH2O | 32.5 ul |
| 总计 | 50 ul |
扩增条件：98℃ 10s，61℃ 30s，72℃ 4.5min，共30个循环。
胶回收I-TevIN201-Cas9null片段，用NdeI和BamHII酶切原核表达载体pET-16b，胶回收骨架载体。两个片段进行In-Fusion反应（Clothech），转化，克隆鉴定，最终获得pET16b- I-TevIN201-Cas9null原核表达载体。
第三部分： I-TevIN201-Cas9null蛋白的表达与纯化
重组质粒pET16b-I-TevIN201-Cas9null转化大肠杆菌E. coli BL21：转化菌在 5ml 氨苄抗性的培养基中培养过夜，次日转移入 500ml 相同的培养基中培养，经IPTG16℃过夜诱导，收集所得到的pET16b-I-TevIN201-Cas9null蛋白菌体，经过高压破碎后离心获得可溶性的上清，进行Ni柱亲和层析。待目的蛋白与Ni柱结合之后，先用漂洗液(20mM Tris-HCl, 500mM NaCl, 5%（v/v）甘油，60mM 咪唑，pH8.0 ) 洗涤，之后用洗脱缓冲液（20mM Tris-HCl, 500mM NaCl, 5%（v/v）甘油，500mM 咪唑，pH8.0 )洗脱掉蛋白 。利用PD-10脱盐柱将洗脱组分中的高盐缓冲液置换成含有20%（体积百分比）甘油的PBS溶液，之后进行SDS-PAGE的检测， 所得即为纯化的I-TevIN201-Cas9null蛋白。
实施例2 I-TevI N201 -Cas9 null 蛋白活性的验证
将I-TevIN201-Cas9null蛋白(300 nM)与线性化的质粒pDNA3.1，tracrRNA，Mg2+，crRNA不同组分孵育。发现TevN201-Cas9null蛋白与线性化的质粒pDNA3.1，tracrRNA，Mg2+，gRNA孵育这一组，DNA有切割活性。说明TevN201-Cas9null蛋白 (300 nM)有活性。
具体如下：体外转录的tracrRNA和crRNA，95°C变性，然后慢慢冷却到室温，将I-TevIN201-Cas9null蛋白与线性化和非线性化的质粒pDNA3.1(200 ng)，Mg2+，tracrRNA (100 nM)，crRNA (100 nM)不同组分37°C孵育一个小时。其中反应的buffer为：20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA，10 mM MgCl2。反应一个小时后，加入5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA)电泳。结果显示如图1所示。
实施例3
如图2、图3、图4所示，在小鼠Srebf2基因上选取I-TevIN201-Cas9null的靶位点（CGCTG TAGTCCGCTGCTCCCTTGGGCCTG），其中CGCTG为Tev识别位点，CCGCTGCTCCCTTGGGCCTG为gRNA序列。I-TevIN201-Cas9null真核表达载体和gRNA载体同时转染小鼠L929细胞（购于中科院），经过Hygromycin筛选，挑取单克隆。放到96孔板培养。等细胞长满后，每孔加入47.5 ul细胞裂解液和2.5 ul 10 mg/ml蛋白酶K，将板置于56 oC孵育箱消化过夜。次日每板配制10 ml预冷无水乙醇+150 ul 5M NaCl的混合液，室温下静置2 hrs，让DNA沉淀，离心，洗脱得到小鼠细胞基因组DNA。提取细胞基因组DNA，用引物primer F2: GCCAGATGCACAGATGAAGCCT（SEQ ID NO.7）和引物Primer R2：AGCCGCTACCATGACATTCCAG（SEQ ID NO.8）进行扩增，配置反应体系和反应程序如下：
表3 反应体系
| 10×Taq Buffer | 5 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq (5 U/μl) | 0.5ul |
| primer F2 20pmol | 1ul |
| Primer R2 20pmol | 1ul |
| 基因组DNA 100ng/ul | 1ul |
| ddH2O | 37.5 ul |
| 总计 | 50 ul |
扩增条件：95℃ 3min，95℃ 10s，60℃ 30s，72℃ 30s，共30个循环。
回收PCR产物送测序，结果显示与野生型对照对比，表达I-TevIN201-Cas9null真核表达载体和gRNA载体的单克隆细胞株，Srebf2基因都有切割。
所述融合蛋白及上述引物的序列如下所示：
SEQ ID NO.1:
MKSGIYQIKN TLNNKVYVGS AKDFEKRWKR HFKDLEKGCH SSIKLQRSFN KHGNVFECSI 60
LEEIPYEKDL IIERENFWIK ELNSKINGYN IADATFGDTC STHPLKEEII KKRSETVKAK 120
MLKLGPDGRK ALYSKPGSKN GRWNPETHKF CKCGVRIQTS AYTCSKCRNR SGENNSFFNH 180
KHSDITKSKI SEKMKGKKPS NDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR 240
HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR 300
LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH 360
MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR 420
RLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA 480
QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR 540
QQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR 600
KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS 660
RFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV 720
YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI 780
SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA 840
HLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD 900
SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV 960
IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR 1020
DMYVDQELDI NRLSDYDVAA IVPQSFLKDD SIDNKVLTRS DKARGKSDNV PSEEVVKKMK 1080
NYWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN 1140
TKYDENDKLI REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK 1200
YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR 1260
PLIETNGETG EIVWDKGRDF ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI 1320
ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID 1380
FLEAKGYKEV KKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS 1440
HYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK 1500
PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI 1560
DLSQLGGDSR ADPKKKRKV 1579
SEQ ID NO.2:
atgaagagcg gcatctacca gatcaagaac accctgaaca acaaggtgta cgtgggcagc 60
gccaaggact tcgagaagcg ctggaagcgc cacttcaagg acctggagaa gggctgccac 120
agcagcatca agctgcagcg cagcttcaac aagcacggca acgtgttcga gtgcagcatc 180
ctggaggaga tcccctacga gaaggacctg atcatcgagc gcgagaactt ctggatcaag 240
gagctgaaca gcaagatcaa cggctacaac atcgccgacg ccaccttcgg cgacacctgc 300
agcacccacc ccctgaagga ggagatcatc aagaagcgca gcgagaccgt gaaggccaag 360
atgctgaagc tgggccccga cggccgcaag gccctgtaca gcaagcccgg cagcaagaac 420
ggccgctgga accccgagac ccacaagttc tgcaagtgcg gcgtgcgcat ccagaccagc 480
gcctacacct gcagcaagtg ccgcaaccgc agcggcgaga acaacagctt cttcaaccac 540
aagcacagcg acatcaccaa gagcaagatc agcgagaaga tgaagggcaa gaagcccagc 600
aacgacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 660
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 720
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 780
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 840
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 900
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 960
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 1020
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 1080
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 1140
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 1200
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 1260
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 1320
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 1380
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 1440
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 1500
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 1560
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1620
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1680
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1740
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1800
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1860
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1920
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1980
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 2040
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 2100
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 2160
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 2220
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 2280
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 2340
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 2400
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 2460
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 2520
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 2580
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2640
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2700
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2760
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2820
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2880
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2940
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 3000
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 3060
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 3120
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 3180
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 3240
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 3300
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 3360
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 3420
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 3480
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 3540
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3600
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3660
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3720
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3780
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3840
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3900
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3960
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 4020
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 4080
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 4140
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 4200
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 4260
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 4320
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 4380
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 4440
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 4500
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 4560
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4620
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4680
gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtga 4740
表4 引物序列
Claims (6)
1.I-TevIN201-Cas9null融合蛋白，其特征在于，所述融合蛋白为由SEQ ID NO.1所示氨基酸序列组成的蛋白。
2.编码权利要求1所述的I-TevIN201-Cas9null融合蛋白的核苷酸序列。
3.根据权利要求2所述的核苷酸序列，其特征在于，所述核苷酸序列如SEQ ID NO.2所示。
4.根据权利要求1所述的I-TevIN201-Cas9null融合蛋白，其特征在于，其第十位天冬氨酸突变成丙氨酸，第839位组氨酸突变成丙氨酸，第840位组氨酸突变成丙氨酸，第863位天冬酰胺突变成丙氨酸，该突变蛋白没有核酸酶活性。
5.根据权利要求1所述的I-TevIN201-Cas9null融合蛋白，其特征在于，其中I-TevIN201蛋白由归巢酶I-TevI N末端201个氨基酸组成，包含I-TevI的酶的催化活性区域和连接域。
6.权利要求1所述的I-TevIN201-Cas9null融合蛋白在药物筛选及基因修饰中的应用。
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