CN107686848A - 转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体及其应用 - Google Patents

转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体及其应用 Download PDF

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CN107686848A
CN107686848A CN201710880749.3A CN201710880749A CN107686848A CN 107686848 A CN107686848 A CN 107686848A CN 201710880749 A CN201710880749 A CN 201710880749A CN 107686848 A CN107686848 A CN 107686848A
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郭雅彬
胡开顺
陈震
李瑜
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Sun Yat Sen Memorial Hospital Sun Yat Sen University
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Abstract

本发明公开一种转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体及其应用，属于基因工程领域。该单质粒载体是含有IRDR‑L‑IRDR‑R盒的双链环状质粒，所述IRDR‑L‑IRDR‑R盒包括IRDR‑L序列、启动子、gRNA scaffold序列、Cas9蛋白序列、抗性筛选基因序列和IRDR‑R序列。本发明的单质粒载体仅需要一次构建、一次转染即可实现sgRNA和Cas9蛋白的表达，该法过程简单、高效、快捷，大大简化质粒构建流程，缩短实验周期，提高工作效率。本发明的载体带有转座酶识别序列，不使用病毒，能方便、快速、安全地建立基因敲除稳定株；包括嘌呤霉素筛选抗性，便于稳定株的筛选。

Description

转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体及其 应用
技术领域
本发明属于基因工程领域，涉及一种单质粒载体，特别涉及一种转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体及其应用。
背景技术
基因功能验证是基因工程的先导，包括基因过表达和基因沉默两种主要方法。CRISPR/Cas9系统是新兴的准确高效的基因敲除系统，该系统借鉴于细菌适应性免疫系统，利用sgRNA和Cas9两个主要元件。其中，sgRNA含有位点特异性靶点序列，引导Cas9核酸酶准确结合到基因组特异位置切割形成DNA双链断裂，生物体损伤修复的不精确性会导致该靶点处基因突变，导致其功能丧失。常规的CRISPR/Cas9系统由两个质粒构建而成，将sgRNA与Cas9蛋白在两个质粒系统分开表达，具有构建过程繁琐、时间长、效率低的缺陷。
转座子(Transposon)亦称跳跃基因或可移动元件，最早由McClintock在研究玉米籽粒的颜色变异中发现。利用“睡美人”(Sleeping Beauty)转座系统作为基因导入手段已在多个技术领域得到广泛应用，该系统利用转座酶SB100X识别转座子两端特异反向重复序列(IRDR)，将转座子序列切出，通过DNA攻击的方式插入新的基因组位置。与传统病毒感染构建稳定细胞株的方法相比，该系统缩短了稳定株构建的时间，避免了包装病毒过程中所产生的生物安全问题，同时提高了稳定重组的效率。
发明内容
为了克服现有技术的缺点与不足，本发明的首要目的在于提供一种转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体。
该载体以更为方便快捷地用于基因组编辑。该单质粒载体仅需一次转染即可完成sgRNA与Cas9蛋白的共同表达，同时载体上融入的Sleeping Beauty转座子元件(IRDR)在转座酶SB100X的作用下，将上述Cas9系统整合受体基因组中，构建出目的基因敲除稳定株。
本发明的另一目的在于提供上述转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体的应用。
本发明的目的通过下述技术方案实现：
本发明提供一种转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体，是含有IRDR-L-IRDR-R盒的双链环状质粒，所述IRDR-L-IRDR-R盒包括IRDR-L序列、启动子、gRNAscaffold序列、Cas9蛋白序列、抗性筛选基因序列和IRDR-R序列。
所述的IRDR-L序列为SEQ ID NO：1中自5′端第11417位～11643位碱基的反向互补序列；所述的IRDR-R序列为SEQ ID NO：1中自5′端第8271位～8498位碱基。
所述的启动子是U6启动子，其序列为SEQ ID NO：1中自5′端第10位～250位碱基。
所述的gRNA scaffold序列为SEQ ID NO：1中自5′端第2140位～2215位碱基。
所述的Cas9蛋白序列为SEQ ID NO：1中自5′端第2513位～6616位碱基。
所述的抗性筛选基因是指嘌呤霉素(puromycin)抗性基因，其序列为SEQ ID NO：1中自5′端第6755位～7351位碱基。
所述的单质粒载体pSM-CRISPR-Puro的核苷酸序列如SEQ ID NO：1所示。
所述的单质粒载体在构建基因敲除稳定株中的应用。
此外本发明还提供了pSM-CRISPR-Puro载体在构建基因敲除稳定株中的方法及应用，包括以下步骤：
(1)利用在线网站(http://crispr.mit.edu/)设计靶向目的基因的guidesequence序列；
(2)选取合适的sgRNA序列，合成两条包含接头的引物；
(3)BsmBI酶切pSM-CRISPR-Puro克隆载体，产物跑胶回收，得酶切回收产物；
(4)引物退火，得退火产物；
(5)连接酶切回收产物与退火产物；
(6)连接产物转化感受态细胞，挑取单克隆，测序获得阳性菌；
(6)阳性菌株扩繁，大提质粒；
(7)将带有目的基因sgRNA序列的pSM-CRISPR-Puro载体与转座酶质粒SB100X共转染宿主细胞，使Cas9系统整合至宿主基因组中；
(8)使用嘌呤霉素筛选得到目的基因敲除的稳转细胞株，扩大培养；
(9)收集培养的细胞，在非变性状态下裂解细胞，离心抽取蛋白，免疫印迹检测(Western Blot)目的基因表达水平；获得目的基因敲除稳定株。
本发明相对于现有技术，具有如下的优点及效果：
(1)本发明的单质粒载体仅需要一次构建、一次转染即可实现sgRNA和Cas9蛋白的表达，该法过程简单、高效、快捷，大大简化质粒构建流程，缩短实验周期，提高工作效率。
(2)本发明的载体带有转座酶识别序列，不使用病毒，能方便、快速、安全地建立基因敲除稳定株。
(3)本发明的质粒载体包括嘌呤霉素(puromycin)筛选抗性，便于稳定株的筛选。
附图说明
图1是pSM-CRISPR-Puro质粒载体结构示意图。
图2是BsmBI核酸内切酶酶切pSM-CRISPR-Puro载体，产物琼脂糖凝胶电泳示意图。
图3是通过免疫印迹检测(Western Blot)的方法检测pSM-CRISPR-Puro-sgRCC2基因敲除稳定株中RCC2蛋白的表达水平，并以微管蛋白(Tubulin)作为内参。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
本发明中若无特别说明，原料均可市购获得，方法为本领域的常规方法。
本发明实施例中所述的pSM-CRISPR-Puro质粒载体结构示意图，如图1所示。
实施例1：
本实施例以RCC2(Gene Bank登录号：BC042141.1)为目标基因，以宫颈癌细胞系HeLa为模型，构建了RCC2基因稳定敲除的HeLa细胞株。
1.1pSM-CRISPR-Puro-sgRCC2质粒载体的构建
(1)登录在线网站(http://crispr.mit.edu/)设计靶向RCC2的sgRNA序列；
(2)选取两对合适的sgRNA序列，加入接头，分别标记为sgRCC2-1、sgRCC2-2，交由华大基因公司合成，其序列如下：
sgRCC2-1-F：5′-CACCGTGCAGTAGCAGCAGCGGCGG-3′；
sgRCC2-1-R：5′-AAACCCGCCGCTGCTGCTACTGCAC-3′；
sgRCC2-2-F：5′-CACCGGCGACAGCAGGCAAGGCGGG-3′；
sgRCC2-2-R：5′-AAACCCCGCCTTGCCTGCTGTCGCC-3′；
(3)BsmBI限制性核酸内切酶37℃酶切pSM-CRISPR-Puro载体30min，酶切体系如表1所示，产物琼脂糖胶电泳，电泳结果如图2所示，回收酶切后的大片段条带；
表1酶切体系
组分 用量
pSM-CRISPR-Puro载体 2μg
BsmBI酶 1μL
10×BsmBI buffer 2μL
ddH2O 补足20μL
总体积 20μL
(4)高温退火合成的引物，程序为：95℃反应5min，0.1℃/s梯度降温至25℃；反应体系如表2所示：
表2引物退火反应体系
组分 用量
sgRCC2-1-F(100μM) 1μL
sgRCC2-1-R(100μM) 1μL
ddH2O 补足20μL
总体积 20μL
或，
组分 用量
sgRCC2-2-F(100μM) 1μL
sgRCC2-2-R(100μM) 1μL
ddH2O 补足20μL
总体积 20μL
(5)22℃连接上述反应得到的胶回收产物与退火产物1h；连接体系如表3所示：
表3连接体系
组分 用量
酶切回收产物 3μL
引物退火产物 10μL
T4DNA ligase 0.5μL
Ligation buffer 2μL
ddH2O 补足20μL
总体积 20μL
(6)取10μL连接产物转化Stbl3感受态细胞(市售)，铺氨苄青霉素(Amp+)抗性LB平板，37℃孵化过夜；
(7)挑取抗性板上单克隆菌株，以U6通用引物测序，筛选出测序含有sgRNA序列的阳性菌株；
(8)扩繁阳性菌株，提取质粒，获得pSM-CRISPR-Puro-sgRCC2质粒载体。
1.2RCC2稳定敲除HeLa细胞株的构建
(1)选取生长状态良好的HeLa细胞，用胰酶消化并计数，吸取200万个细胞种板至6cm细胞培养皿中；
(2)第二天待细胞完全贴壁后，使用lipofectamine 2000共转染5μg pSM-CRISPR-Puro-sgRCC2和5μg SB100X转座酶质粒于HeLa细胞中；
(3)转染后36小时，使用含有终浓度为2μg/mL嘌呤霉素的培养基筛选稳定株，筛选4天左右可获得RCC2稳定敲除HeLa细胞株。
1.3免疫印迹检测RCC2基因表达水平
收取一部分细胞，RIPA裂解抽取蛋白，经免疫印迹(Western Blot)验证，结果如图3所示：该细胞系RCC2基因稳定敲除。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
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<110> 中山大学孙逸仙纪念医院
<120> 转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体及其应用
<130> 1
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11643
<212> DNA
<213> Artificial Sequence
<220>
<221> allele
<222> (1)..(11643)
<223> 单质粒载体pSM-CRISPR-Puro的核苷酸序列
<400> 1
taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 60
gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg 120
tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 180
gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 240
tggaaaggac gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa 300
tattatattc tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat 360
caataattag aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa 420
aaaacgctag ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc 480
acttgttcct aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct 540
aactccacca gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac 600
atcttcaaat gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc 660
agcttcaaca gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc 720
cataatacat ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa 780
aactaattca taatttttgt cttttaatgc accaatattt aataccatat caatttctgt 840
tgcaccatct ttaattgctt cagaaacttc gaatgctttt gtagctgttg tgcatgcacc 900
tagaggaaaa cctacaacat ttgttattcc tacatttgtg ccttttaata attctttaca 960
atagcttgtt caatatgaat taacacaaac tgttgcaaaa tcaaattcaa ttgcttcatc 1020
acataattgt ttaatttcag ctttcgtagc atcttgtttt aataatgtgt gatctatata 1080
tttgtttagt ttcatttttt ctcctatata ttcattttta attttaattc tttaataatt 1140
tcgtctactt taactttagc gttttgaaca gattcaccaa cacctataaa ataaattttt 1200
agtttaggtt cagttccact tgggcgaaca gcaaatcatg acttatcttc taaataaaat 1260
tttagtaagt cttgtcctgg catattatac attccatcga tgtagtcttc aacattaaca 1320
actttaagtc cagcaatttg agttaagggt gttgctctca atgatttcat taatggttca 1380
atttttaatt tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca 1440
cccatttctt taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa 1500
aatcaagcag cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag 1560
tcatcaatta catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct 1620
tcttctttag caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta 1680
actccatatt tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat 1740
agagccggat tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt 1800
aaaattggtc ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc 1860
attccaaatc tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata 1920
ccatattcaa gcggtatttt tcatgcagga tcaaattctg gatttggatt tacaacattt 1980
ttaaatgttt catcttcaaa tgcatgctct tcaacctcaa taacgttata tcctgattca 2040
cgtaatattt ttggggtaaa tttagttcct gttccattaa ctgcgctaaa aataattttt 2100
aaatcttttt tagcttcttg ctcttttttg tacgtctctg ttttagagct agaaatagca 2160
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 2220
tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc 2280
agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg 2340
tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct 2400
ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt 2460
tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa 2520
gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga 2580
cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 2640
caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 2700
gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 2760
agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 2820
gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 2880
ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 2940
ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 3000
gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 3060
gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 3120
cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 3180
aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc 3240
cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa 3300
actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 3360
cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 3420
cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 3480
gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 3540
gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 3600
tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 3660
ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 3720
gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct 3780
gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 3840
cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 3900
ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga 3960
caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 4020
caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 4080
gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 4140
gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 4200
gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 4260
ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 4320
caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 4380
gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 4440
cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 4500
ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 4560
gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac 4620
ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 4680
cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 4740
ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 4800
ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 4860
gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 4920
cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 4980
cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc 5040
tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 5100
ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 5160
gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 5220
cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 5280
aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 5340
cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 5400
gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 5460
cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 5520
gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 5580
caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 5640
gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 5700
cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 5760
gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 5820
cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 5880
gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 5940
gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 6000
gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 6060
agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 6120
gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg 6180
aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 6240
gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 6300
gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 6360
cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 6420
agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 6480
cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 6540
ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 6600
tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa 6660
gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa 6720
acaagccgga gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct 6780
cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta 6840
ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca 6900
agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg 6960
cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga 7020
gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga 7080
aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt 7140
ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc 7200
ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt 7260
ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac 7320
ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga 7380
ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 7440
tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 7500
ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 7560
gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 7620
caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 7680
actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 7740
ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 7800
ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 7860
tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 7920
gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac 7980
aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt 8040
cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag 8100
atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 8160
ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 8220
tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt ttgagtgtat 8280
gtaaacttct gacccactgg gaatgtgatg aaagaaataa aagctgaaat gaatcattct 8340
ctctactatt attctgatat ttcacattct taaaataaag tggtgatcct aactgaccta 8400
agacagggaa tttttactag gattaaatgt caggaattgt gaaaaagtga gtttaaatgt 8460
atttggctaa ggtgtatgta aacttccgac ttcaactgta tagggatcct ctagctagag 8520
tcgacctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg gttaatttcg 8580
agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 8640
ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 8700
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 8760
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 8820
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 8880
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 8940
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 9000
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 9060
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 9120
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 9180
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 9240
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 9300
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 9360
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 9420
aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 9480
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 9540
ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 9600
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 9660
atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 9720
tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 9780
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 9840
tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 9900
gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 9960
cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 10020
gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 10080
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 10140
aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 10200
atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 10260
aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 10320
aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 10380
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 10440
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 10500
gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 10560
ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 10620
ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 10680
atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 10740
gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 10800
agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 10860
tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 10920
ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 10980
cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 11040
tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 11100
tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 11160
caaaaattta acgcgaattt taacaaaata ttaacgctta caatttccat tcgccattca 11220
ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg 11280
cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 11340
gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg 11400
agctcggatc cctatacagt tgaagtcgga agtttacata cacttaagtt ggagtcatta 11460
aaactcgttt ttcaactact ccacaaattt cttgttaaca aacaatagtt ttggcaagtc 11520
agttaggaca tctactttgt gcatgacaca agtcattttt ccaacaattg tttacagaca 11580
gattatttca cttataattc actgtatcac aattccagtg ggtcagaagt ttacatacac 11640
taa 11643
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<221> allele
<222> (1)..(25)
<223> sgRCC2-1-F
<400> 2
caccgtgcag tagcagcagc ggcgg 25
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<221> allele
<222> (1)..(25)
<223> sgRCC2-1-R
<400> 3
aaacccgccg ctgctgctac tgcac 25
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<221> allele
<222> (1)..(25)
<223> sgRCC2-2-F
<400> 4
caccggcgac agcaggcaag gcggg 25
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<221> allele
<222> (1)..(25)
<223> sgRCC2-2-R
<400> 5
aaaccccgcc ttgcctgctg tcgcc 25

Claims (10)

1.一种转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体，其特征在于：
所述的单质粒载体是含有IRDR-L-IRDR-R盒的双链环状质粒，所述IRDR-L-IRDR-R盒包括IRDR-L序列、启动子、gRNA scaffold序列、Cas9蛋白序列、抗性筛选基因序列和IRDR-R序列。
2.根据权利要求1所述的转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体，其特征在于：
所述的IRDR-L序列为SEQ ID NO：1中自5′端第11417位～11643位碱基的反向互补序列；
所述的IRDR-R序列为SEQ ID NO：1中自5′端第8271位～8498位碱基。
3.根据权利要求1所述的转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体，其特征在于：
所述的启动子是U6启动子。
4.根据权利要求3所述的转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体，其特征在于：
所述的U6启动子的序列为SEQ ID NO：1中自5′端第10位～250位碱基。
5.根据权利要求1所述的转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体，其特征在于：
所述的gRNA scaffold序列为SEQ ID NO：1中自5′端第2140位～2215位碱基。
6.根据权利要求1所述的转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体，其特征在于：
所述的Cas9蛋白序列为SEQ ID NO：1中自5′端第2513位～6616位碱基。
7.根据权利要求1所述的转座子协同CRISPR/Cas9系统的稳定敲除单质粒载体，其特征在于：
所述的抗性筛选基因是指嘌呤霉素抗性基因。
8.一种单质粒载体pSM-CRISPR-Puro，其特征在于：所述的单质粒载体pSM-CRISPR-Puro的核苷酸序列如SEQ ID NO：1所示。
9.权利要求1～7任一项所述的稳定敲除单质粒载体或权利要求8所述的单质粒载体pSM-CRISPR-Puro在构建基因敲除稳定株中的应用。
10.一种构建基因敲除稳定株的方法，其特征在于包括如下步骤：
(1)设计靶向目的基因的guide sequence序列；
(2)选取合适的sgRNA序列，合成两条包含接头的引物；
(3)BsmBI酶切权利要求1～7任一项所述的稳定敲除单质粒载体或权利要求8所述的单质粒载体pSM-CRISPR-Puro，产物跑胶回收，得酶切回收产物；
(4)引物退火，得退火产物；
(5)连接酶切回收产物与退火产物；
(6)连接产物转化感受态细胞，挑取单克隆，测序获得阳性菌；
(6)阳性菌株扩繁，大提质粒；
(7)将带有目的基因sgRNA序列的单质粒载体与转座酶质粒SB100X共转染宿主细胞，使Cas9系统整合至宿主基因组中；
(8)使用抗性筛选基因筛选得到目的基因敲除的稳转细胞株，扩大培养；
(9)收集培养的细胞，在非变性状态下裂解细胞，离心抽取蛋白，免疫印迹检测目的基因表达水平；获得目的基因敲除稳定株。
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US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
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