CN106191071A - 一种CRISPR‑Cas9系统及其用于治疗乳腺癌疾病的应用 - Google Patents

一种CRISPR‑Cas9系统及其用于治疗乳腺癌疾病的应用 Download PDF

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CN106191071A
CN106191071A CN201610705832.2A CN201610705832A CN106191071A CN 106191071 A CN106191071 A CN 106191071A CN 201610705832 A CN201610705832 A CN 201610705832A CN 106191071 A CN106191071 A CN 106191071A
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李蒙
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Abstract

本发明提供了一种CRISPR‑Cas9系统及其用于治疗乳腺癌疾病的应用，其中提供了多个sgRNA可以实现RecQL4基因的高效敲除。相比于ZFNs和TALENs的复杂表达结构，siRNA的使用复杂以及效率低下，CRISPR‑Cas9系统中的Cas9表达结构是固定不变的，针对不同基因只需要将的识别序列插入sgRNA表达结构中即可完成系统组建，操作简单，成本低，适用于大规模的哺乳动物基因打靶工作。

Description

一种CRISPR-Cas9系统及其用于治疗乳腺癌疾病的应用
技术领域
本发明涉及基因工程领域，更具体地说涉及CRISPR-Cas9特异性敲除人RecQL4基因的方法以及用于特异性靶向RecQL4基因的sgRNA。
背景技术
乳腺癌是女性最常见的癌症，主要包括导管癌和小叶癌。2011年美国CA(A CancerJournal化r Clinicians)公布的最新统计数据显示，美国2011年预计将有230480例女性患乳腺癌，占女性新发恶性肿瘤的30％，居女性恶性肿瘤发病率第一位，并且死亡人数将达39520例。更为严重的是全球的乳腺癌发病率正在逐年增长。乳腺癌的诊断方法主要包括乳腺钢祀X线摄影、乳腺彩色多谱勒超声检查、乳腺导管内视镜检查、乳腺导管灌洗、CT和磁共振。但是该些检测方法主要是基于形态学标准，依然缺乏与乳腺癌相关的特异性标志。乳腺癌的治疗主要包括手术切除、化疗及放疗或者几者综合治疗等方式。但是传统化疗、放疗的方式由于缺乏肿瘤细胞的祀向性导致非常严重的副作用。近年来发展的单克隆抗体虽然具有较高的特异性和亲和力，但是抗体本身所具有的一些缺陷，如免疫原性、不稳定性、批次间的差异性等使其在临床上的实际应用中具有一定的局限性。因此，找到乳腺肿瘤细胞的特异性祀标，在肿瘤的诊断和治疗中都具有重要意义。近些年发展的核酸适体探针，尤其是细胞祀向的核酸适体，能够特异性识别包括全血样品的复杂样品中的癌细胞，将给肿瘤的早期特异性快速诊断和祀向治疗带来新的希望和解决途径。目前没有用于治疗或预防乳腺癌的普遍成功的方法。
目前乳腺癌的治疗依赖于早期诊断(例如，通过常规的乳腺筛查方法)和侵入性治疗的组合，其可包括多种治疗例如手术、放射疗法、化学疗法和激素疗法中的一种或多种。通常基于许多预后参数(包括特异性肿瘤标记的分析)来选择特定乳腺癌的疗程。
RecQL4是RecQ解旋酶家族中的一员。它是由Kitao等人在1998年克隆并以大肠杆菌(E.Coli)RecQ蛋白来命名的。其主要功能是打开DNA双螺旋结构用于DNA转录、复制以及损伤修复(见Genetics in Medicine,2006,The versatile RecQL4,8(4):213-216)，因此，RecQL4对于维持基因组的稳定性是非常重要的。而基因的不稳定性在肿瘤发生及发展中起重要作用。RecQL4功能缺失可引起人类基因组不稳定性疾病＝Rothmund-Thomson综合症(RTQ，表现为皮肤红斑、骨骼异常、早衰和肿瘤特别是骨肉瘤高发。除了RTS，RecQL4功能缺失也与RAPADILINO和BALLER-GEROLD综合症相关。这三种临床疾病症候群的共同特征是个矮和骨骼异常，然而肿瘤高发只存在于RTS及RAPADILINO疾病症候群。使用RecQL4基因敲除小鼠模型进一步证实了在人类中的发现。小鼠在RecQL4基因功能缺失之后，表现为生长迟缓、早死、以及其它的类似于人类Rothmund-Thomson综合症的缺陷。
CN 101476163 A中公开了抑制人RecQL4基因表达的siRNA能够用于抗乳腺肿瘤。但是siRNA的使用具有药物的递送效率低，要持续给药，并不能够彻底根治，而且用药复杂，不适宜大规模推广。
CRISPR是一个特殊的DNA重复序列家族，广泛分布于细菌和古细菌基因组中。CRISPR位点通常由短的高度保守的重复序列(repeats)组成，重复序列的长度通常21～48bp，重复序列之间被26～72bp间隔序列(spacer)隔开。CRISPR就是通过这些间隔序列与靶基因进行识别。Cas(CRISPRassociated)存在于
CRISPR位点附近，是一种双链DNA核酸酶，能在导向RNA(guideRNA，gRNA)的引导下对革EU立点进行切割。它与folk酶功能类似，但是它并不需要形成二聚体才能发挥作用。
这一技术由于能快速、简便、高效地靶向基因组任何基因，从而引起了广泛的关注，在2012年开始像爆炸一般流行开来。由于其容易操作、可以同时靶向多个基因，可以高通量制备、造价低等优势，Cas9已经成为一种发展最快的技术(Pennisi，2013)。正是由于其优越性，这一技术在Nature推荐的2013十大进展中位列第一。
Cas9靶向切割DNA是通过两种小RNA--crRNA(CRISPR RNA)和tracrRNA(trans-activating crRNA)和革E序列互补识别的原理实现的。现在已经把两种小RNA融合成一条RNA链，简称sgRNA(single guide RNA)。因此，sgRNA能否做到特异性、精确祀向目标基因是CRISPR-Cas9能否特异性敲除目标基因的先决条件，无论是脱靶还是错误靶向，都会影响CRISPR-Cas9对目标基因的特异性敲除。因此，能够设计、制备出精确性和特异性靶向目标基因的sgRNA成为CRISPR-Cas9基因敲除的关键技术。
与ZFN相比，CRISPR-Cas9具有更快速、简便、高效、多位点、特异性靶向敲除基因的优势。为高效靶向敲除RecQL4,实现艾滋病及其相关疾病的治疗提供了一种可能的选择。本发明的目的就是要验证利用CRISPR-Cas9高效靶向敲除RecQL4提供相应的技术方案，以达到特异性敲除RecQL4的目的。
发明内容
针对现有使用siRNA靶向RecQL4进行乳腺癌疾病治疗所存在的问题：为了解决上述技术问题，本申请的技术方案如下:
一、sgRNA寡核普酸的设计和选择
1.靶向RecQL4基因的sgRNA的设计:
因为没有使用体外转录，只是构建普通载体的方式制作。所以如无特殊说明，文中的sgRNA序列指的是sgRNA对应DNA序列。
首先在Z38基因上选择5’-GGN(19)GG的序列，如果没有5’-GGN(19)GG的序列，5’-GN(20)GG或者5’-N(21)GG也可以。sgRNA在STAT6基因上的靶向位点位于基因的外显子。在UCSC数据库中用BLAT或NCBI数据库中用BLAST，确定sgRNA的靶序列是否唯一。同时根据其他的sgRNA的设计规则，共设计了57个sgRNA，然而通过最后的实验证实，其中只有17个具有靶向修饰的功能，在此，没有功能的序列就不一一列举，仅仅给出2个反例，这也充分说明在现有技术中，sgRNA的设计并不不能仅仅根据设计规则不需要实验即可得到有功能的sgRNA，本发明涉及的sgRNA序列如下：
RecQL4-sg1：gcaagcgcggaggccgggcgggcg(SEQ ID NO:2)
RecQL4-sg2：ggcgtgggagcgcgcgttccgac(SEQ ID NO:3)
RecQL4-sg3：acgtggaggcggcgccggaggaga(SEQ ID NO:4)
RecQL4-sg4：cagcggctcaaggccaatctgaaa(SEQ ID NO:5)
RecQL4-sg5：ccaaggccaggccggctccagc(SEQ ID NO:6)
RecQL4-sg6：cagtgaggtcccagattttctggg(SEQ ID NO:7)
RecQL4-sg7：aacttctgatccctggtgagtc(SEQ ID NO:8)
RecQL4-sg8：agtggaggcgagaagcggagat(SEQ ID NO:9)
RecQL4-sg9：gagccctgggagagccccgcaca(SEQ ID NO:10)
RecQL4-sg10：caagctagggctgggaaggctga(SEQ ID NO:11)
RecQL4-sg11：ctactccctggggccctcagggcag(SEQ ID NO:12)
RecQL4-sg12：gccggctgaggtgttccaggccct(SEQ ID NO:13)
RecQL4-sg13：gtgcagtcatgcggatcctgtct(SEQ ID NO:14)
RecQL4-sg14：actcatggatgaccaggtgtct(SEQ ID NO:15)
RecQL4-sg15：caaggcggcctgcatacactc(SEQ ID NO:16)
RecQL4-sg16：gcaaccggcgcgaggacacagagc(SEQ ID NO:17)
RecQL4-sg17：ggcctttgggatggggctggacc(SEQ ID NO:18)
RecQL4-sg18：gcggcggccgaagaggcgccagag(SEQ ID NO:19)
RecQL4-sg19：gcaggcctgatctaggctcaga(SEQ ID NO:20)
二、构建sgRNA的寡聚核苷酸双链
根据选择的sgRNA,在其5’加上CCGG得到正向寡核苷酸(Forward oligo)(如果序列本身在5’端已经有1或者2个G，那么就对应的省略1或者2个G)；根据选择的sgRNA，获得其对应DNA的互补链，并且在其5’加上AAAC得到反向寡核苷酸(Reverse oligo)。分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸的forward oligo和reverseoligo成对变性、退火,退火之后形成可以连入U6真核表达载体的双链,如下:
针对RecQL4-sg1：gcaagcgcggaggccgggcgggcg，设计的正向寡核苷酸(Forwardoligo)为：ccggcaagcgcggaggccgggcgggcg(SEQ ID NO：21)；反向寡核苷酸(Reverseoligo)为aaacgcccgcccggcctccgcgcttgc(SEQ ID NO：22)。
三、sgRNA寡聚核苷酸质粒的构建
1.线性化pGL3-U6-sgRNA质粒。
2.将退火的sgRNA寡聚核普酸双链与线性化pGL3-U6-sgRNA质粒连接获得pGL3-U6-RecQL4-sg1质粒。
3.转化并涂Amp+平板(50微克/ml)。
4.用通用引物U6测序的方法鉴定阳性克隆，所述引物为
atggactatcatatgcttaccgta。
5.37度摇床摇菌过夜并用质粒抽提试剂盒抽提pGL3-U6-RecQL4sg质粒。
四、转染细胞获得RecQL4基因敲除细胞
1、按照LipofectamineTM2000Transfection Reagent(Invitrogen,11668-019)的操作手册，将分别带有对应sgRNA寡聚核苷酸的pGL3-U6-RecQL4-sg1质粒与pST1374-NLS-flag-Cas9-ZF质粒混匀，共转染细胞。
2、用T7EN1酶切检测和TA克隆测序确认RecQL4基因己经被敲除。
有益的效果
本发明利用CRISPR_Cas9系统进行哺乳动物基因打靶，其优点是：
l、sgRNA特异性识别DNA序列中的NGG三个碱基对，识别规则简单且易分析，在靶基因中可以同时找到多个sgRNA识别位点，从而可以根据打靶要求进行选择，适用性广泛；
2、相比于ZFNs和TALENs的复杂表达结构，siRNA的使用复杂以及效率低下，CRISPR-Cas9系统中的Cas9表达结构是固定不变的，针对不同基因只需要将的识别序列插入sgRNA表达结构中即可完成系统组建，操作简单，成本低，适用于大规模的哺乳动物基因打靶工作；
3、利用CRISPR-Cas9系统继续细胞转染时，由于sgRNA表达结构很短，所以可以大大提高转染效率，也可以将Cas9和sgRNA表达结构整合到一个载体中，进一步提高转染效率，这都是ZFNs和TALENs很难做到的；
4、针对同一家族的不同基因或不同物种的同一基因进行基因打靶时，可以选择基因中的保守区进行sgRNA设计，从而实现同一条sgRNA对多个基因的打靶或修饰，相比其他技术更加简便、高效；
5、可以通过向哺乳动物中同时导入一个Cas9表达结构和多个sgRNA的方式轻松实现多基因同时打靶，无论从打靶效率还是操作简便程度上都是其他技术无法比拟的；
6、使用CRISPR打靶，能够从根源性上将基因进行敲除，从而使得治疗效果更稳定。
具体实施方式
下面结合附图和具体的实施例对本发明的技术方案做进一步介绍。
实施例1sgRNA的设计
首先根据SEQ ID NO：1所示的RecQL4基因上选择5’-GGN(19)GG的序列，如果没有5’-GGN(19)GG的序列，5’-GN(20)GG或者5’-N(21)GG也可以。sgRNA在STAT6基因上的靶向位点位于基因的外显子。在UCSC数据库中用BLAT或NCBI数据库中用BLAST，确定sgRNA的靶序列是否唯一。同时根据其他的sgRNA的设计规则，共设计了57个sgRNA，然而通过最后的实验证实，其中只有17个具有靶向修饰的功能，在此，没有功能的序列就不一一列举，仅仅给出2个反例，这也充分说明在现有技术中，sgRNA的设计并不不能仅仅根据设计规则不需要实验即可得到有功能的sgRNA，本发明涉及的sgRNA序列如下：
RecQL4-sg1：gcaagcgcggaggccgggcgggcg(SEQ ID NO:2)
RecQL4-sg2：ggcgtgggagcgcgcgttccgac(SEQ ID NO:3)
RecQL4-sg3：acgtggaggcggcgccggaggaga(SEQ ID NO:4)
RecQL4-sg4：cagcggctcaaggccaatctgaaa(SEQ ID NO:5)
RecQL4-sg5：ccaaggccaggccggctccagc(SEQ ID NO:6)
RecQL4-sg6：cagtgaggtcccagattttctggg(SEQ ID NO:7)
RecQL4-sg7：aacttctgatccctggtgagtc(SEQ ID NO:8)
RecQL4-sg8：agtggaggcgagaagcggagat(SEQ ID NO:9)
RecQL4-sg9：gagccctgggagagccccgcaca(SEQ ID NO:10)
RecQL4-sg10：caagctagggctgggaaggctga(SEQ ID NO:11)
RecQL4-sg11：ctactccctggggccctcagggcag(SEQ ID NO:12)
RecQL4-sg12：gccggctgaggtgttccaggccct(SEQ ID NO:13)
RecQL4-sg13：gtgcagtcatgcggatcctgtct(SEQ ID NO:14)
RecQL4-sg14：actcatggatgaccaggtgtct(SEQ ID NO:15)
RecQL4-sg15：caaggcggcctgcatacactc(SEQ ID NO:16)
RecQL4-sg16：gcaaccggcgcgaggacacagagc(SEQ ID NO:17)
RecQL4-sg17：ggcctttgggatggggctggacc(SEQ ID NO:18)
RecQL4-sg18：gcggcggccgaagaggcgccagag(SEQ ID NO:19)
RecQL4-sg19：gcaggcctgatctaggctcaga(SEQ ID NO:20)
实施例2、构建sgRNA的寡聚核苷酸双链
根据选择的sgRNA：RecQL4-sg1,在其5’加上CCGG得到正向寡核苷酸
(Forward oligo)(如果序列本身在5’端已经有1或者2个G，那么就对应的省略1或者2个G)；根据选择的sgRNA，获得其对应DNA的互补链，并且在其5’加上AAAC得到反向寡核苷酸(Reverse oligo)。分别合成上述正向寡核苷酸和反向寡核苷酸，设计的正向寡核苷酸(Forward oligo)为：
ccggcaagcgcggaggccgggcgggcg(SEQ ID NO：21)；反向寡核苷酸(Reverse oligo)为aaacgcccgcccggcctccgcgcttgc(SEQ ID NO：22)。
将合成的sgRNA寡聚核苷酸的forward oligo和reverse oligo成对变性、退火,退火之后形成可以连入U6真核表达载体的双链,如下:
所述条件为：2.5μl forward Oligo(100μM)，2.5μl reverse Oligo(100μM)，1u1NEB buffer，4μl灭菌水。在PCR仪中按照以下touch down程序运行:95度,5min；95-83度at-1.8度/s；85-24度at-0.1度/s；hold at4度。
实施例3、sgRNA寡聚核苷酸质粒的构建
1.线性化pGL3-U6-sgRNA质粒。酶切体系和条件如下:2μg pGL3-U6-sgRNA(400ng/u 1)；1μ1CutSmart Buffer；1μ1BsaI(NEB,R0535L)；补水至50μ1,37度孵育3-4小时，每隔一段时间振荡一下并离心以防液滴蒸发至管盖上。
2.将退火的sgRNA寡聚核苷酸双链与线性化pGL3-U6-sgRNA质粒连接获得pGL3-U6-RecQL4-sg1质粒。
3.转化并涂Amp+平板(50微克/ml)。
4.用通用引物U6测序的方法鉴定阳性克隆，所述引物为
atggactatcatatgcttaccgta。
5.37度摇床摇菌过夜并用质粒抽提试剂盒抽提pGL3-U6-RecQL4-sg1质粒。
实施例4、转染细胞获得RecQL4基因敲除细胞
(1)乳腺癌细胞T47D接种培养于DMEM高糖培养液中，其中含10％FBS，penicillin(100U/ml)和streptomycin(100微克/ml)。(2)在转染前分至12孔板中，待80％密度时进行转染。按照LipofectamineTM2000Transfection Reagent(Invitrogen,11668-019)的操作手册，将分别带有对应sgRNA寡聚核苷酸的pGL3-U6-RecQL4-sg1质粒1微克与pST1374-NLS-flag-Cas9-ZF质粒2微克混匀，共转染至每孔细胞中，6.5h换液，加入Blasticidin和Puromycin药筛，48小时后收集细胞。
T7EN1酶切检测
将收集的细胞在裂解液(10u M Tris-HCl,0.4M NaCl,2u M EDTA,1％SDS)中用100μg/ml蛋白酶K裂解消化后，酚-氯仿抽提后溶解到50u 1去离子水中。(2)使用引物，上游序列为：ctggacgatcgcaagcgcgg，下游引物为：gcacgtagctctcgaagctt(2440bp扩增大小)进行PCR扩增，纯化获得PCR回收产物，取200ng统一稀释到20u 1,在20u 1体系中加入T7EN10.3u 1,37 0C酶切30分钟后，加入2u 1lOXLoading Buffer，用2.5％的琼脂糖胶电泳检测。结果显示，通过琼脂糖胶电泳可以发现:发生断裂末端连接修复的基因组因为与原基因组不完全匹配，而被T7EN1切割。显示出较小的条带。说明基因敲除成功。
将上述步骤获得的PCR回收产物用rTaq进行加A反应。加A反应体系为:800ng PCR回收产物，5u 1lOX Buffer(Mg2+free)，3u 1Mg2+，4u 1dNTP，0.5u 1rTaq(TAKARA,ROOlAM)，补水至50u 1体系。
37度温育30分钟后，取lul产物与pMD19-T vector连接并转化感受态细胞DH5a。挑取单克隆以通用引物U6序列atggactatcatatgcttaccgta测序，根据测序结果发现:靶基因RecQL4缺失了sgRNA靶序列，基因敲除成功。
实施例5
将乳腺癌细胞T47D细胞敲除前后的各取1X 106细胞，收获后，用新鲜加入了各种抑制剂(1mM Na orthovanadate,1mMPMSF,10μg/ml Aprotinin,
Leupeptin,胃酶抑素)的2Oμl裂解液(5OmMHEPES[pΗ7.0],1％NP-40,5mM EDTA,450mM NaCl,IOmM Na pyrophosphate和50mM NaF)在室温下超声处理后，加入I％β巯基乙醇，100℃中放置5分钟。在SDS-PAGE胶中，每孔上样10μ1样品。电泳后转膜将蛋白样品转移到硝酸纤维素膜上。转膜结束后用TTBS洗膜一次，用5％脱脂奶粉封闭膜1小时，用TTBS洗膜一次，将稀释后的一抗与膜室温杂交2小时或4℃过夜。用TTBS洗涤三次后将稀释后的二抗与膜室温杂交1小时，用TTBS洗涤后加入底物显色，暗室曝光，其中以actin作为内参蛋白对照。
结果显示，敲除了RecQL4基因的细胞中的RecQL4蛋白的表达相对于原始细胞，蛋白的表达量显著下降95.8％，这也充分的说明，通过敲除RecQL4的目的已经达到。
实施例6其它sgRNA效果验证
sgRNA选择RecQL4-sg2～19，按照实施例2-5相同的实验方法进行相应的基因敲除以及蛋白水平检测，在此，由于步骤基本相同，具体的操作条件就不一一赘述。通过实验发现，STAT6-sg2～17，这16个sgRNA可以实现基因的敲除，其敲除效率达到了88％以上，而RecQL4-sg18、RecQL4-sg19都没有实现基因的敲除，这也说明sgRNA的选择并非简单容易的。
另外蛋白表达水平检测的结果如下：
从以上结果可以看出，RecQL4-sg1～17都能够实现基因的敲除，并且都能够达到相似的降低RecQL4蛋白表达的效果。
实施例7敲除后细胞的肿瘤致病性验证
将RecQL4基因敲除前后的乳腺癌细胞T47D细胞进行小鼠致病性检测：以每只小鼠200μI PBS中200万个细胞的剂量，经皮下注射入基因分别敲除前后的小鼠中，每个敲除后的细胞设置10个重复。通过每周测量肿瘤大小、体重和生物光学成像来监测癌症生长、侵入和转移。8周后，检测结果如下：
从以上结果可以看出，敲除了RecQL4的癌细胞在致病性上得到了显著的降低。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
〈110〉李蒙
〈120〉一种CRISPR-Cas9系统及其用于治疗乳腺癌疾病的应用
〈160〉22
〈210〉1
〈211〉3840
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4
1 ctggacgatc gcaagcgcgg aggccgggcg ggcgcgcgcg ccatggagcg gctgcgggac
61 gtgcgggagc ggctgcaggc gtgggagcgc gcgttccgac ggcagcgcgg gcggcgaccg
121 agccaggacg acgtggaggc ggcgccggag gagacccgcg cgctctaccg ggaataccgc
181 actctgaagc gtaccacggg ccaggccggc ggcgggctcc gcagctccga gtcgctcccc
241 gcggcggccg aagaggcgcc agagccccgc tgctgggggc cccatctgaa tcgggctgcg
301 accaagagtc cacagcctac gccagggcgg agccgccagg gctcggtgcc ggactacggg
361 cagcggctca aggccaatct gaaaggcacc ctgcaggccg gaccagccct gggccgcaga
421 ccgtggcctc taggaagagc ctcatctaag gcatccaccc caaagccccc aggtacaggg
481 cctgtcccct cctttgcaga aaaagtcagt gatgagcctc cacagctccc tgagccccag
541 ccaaggccag gccggctcca gcatctgcag gcatccctga gccagcggct gggctcccta
601 gatcctggct ggttacagcg atgtcacagt gaggtcccag attttctggg ggcccccaaa
661 gcctgcaggc ctgatctagg ctcagaggaa tcacaacttc tgatccctgg tgagtcggct
721 gtccttggtc ctggtgctgg ctcccagggc ccagaggctt cagccttcca agaagtcagc
781 atccgtgtgg ggagccccca gcccagcagc agtggaggcg agaagcggag atggaacgag
841 gagccctggg agagccccgc acaggtccag caggagagca gccaagctgg acccccatcg
901 gagggggctg gggctgtagc agttgaggaa gaccctccag gggaacctgt acaggcacag
961 ccacctcagc cctgcagcag cccatcgaac cccaggtacc acggactcag cccctccagt
1021 caagctaggg ctgggaaggc tgagggcaca gcccccctgc acatcttccc tcggctggcc
1081 cgccatgaca ggggcaatta cgtacggctc aacatgaagc agaaacacta cgtgcggggc
1141 cgggcactcc gtagcaggct cctccgcaag caggcatgga agcagaagtg gcggaagaaa
1201 ggggagtgtt ttgggggtgg tggtgccaca gtcacaacca aggagtcttg tttcctgaac
1261 gagcagttcg atcactgggc agcccagtgt ccccggccag caagtgagga agacacagat
1321 gctgttgggc ctgagccact ggttccttca ccacaacctg tacctgaggt gcccagcctg
1381 gaccccaccg tgctgccact ctactccctg gggccctcag ggcagttggc agagacgccg
1441 gctgaggtgt tccaggccct ggagcagctg gggcaccaag cctttcgccc tgggcaggag
1501 cgtgcagtca tgcggatcct gtctggcatc tccacgctgc tggtgctgcc tacaggtgcc
1561 ggcaagtccc tgtgctacca gctcccagcg ctgctctaca gccggcgcag cccctgcctc
1621 acgttggtcg tctctcccct gctgtcactc atggatgacc aggtgtctgg cctgccaccg
1681 tgtctcaagg cggcctgcat acactcgggc atgaccagga agcaacggga atctgtcctg
1741 cagaagattc gggcagccca ggtacacgtg ctgatgctga cacctgaggc actggtgggg
1801 gcgggaggcc tccctccagc cgcacagctg cctccagttg cttttgcctg cattgatgag
1861 gcccactgcc tctcccagtg gtcccacaac ttccggccct gctacctgcg cgtctgcaag
1921 gtgcttcggg agcgcatggg cgtgcactgc ttcctgggcc tcacagccac agccacacgc
1981 cgcactgcca gtgacgtggc acagcacctg gctgtggctg aagagcctga cctccacggg
2041 ccagccccag ttcccaccaa cctgcacctt tccgtgtcca tggacaggga cacagaccag
2101 gcactgttga cgctgctgca aggcaaacgt tttcaaaacc tcgattccat tatcatttac
2161 tgcaaccggc gcgaggacac agagcggatc gctgcgctcc tccgaacctg cctgcacgca
2221 gcctgggtcc cagggtctgg aggtcgtgcc cccaaaacca cagccgaggc ctaccacgcg
2281 ggcatgtgca gccgggaacg gcggcgggta cagcgagcct tcatgcaggg ccagttgcgg
2341 gtggtggtgg ccacggtggc ctttgggatg gggctggacc ggccagatgt gcgggctgtg
2401 ctgcatctgg ggctgccccc aagcttcgag agctacgtgc aggccgtggg ccgggccggg
2461 cgtgacgggc agcctgccca ctgccacctc ttcctgcagc cccagggcga agacctgcga
2521 gagctgcgca gacatgtgca cgccgacagc acggacttcc tggctgtgaa gaggctggta
2581 cagcgcgtgt tcccagcctg cacctgcacc tgcaccaggc cgccctcgga gcaggaaggg
2641 gccgtgggtg gggagaggcc tgtgcccaag tacccccctc aagaggctga gcagcttagc
2701 caccaagcag ccccaggacc cagaagggtc tgcatgggcc atgagcgggc actcccaata
2761 cagcttaccg tacaggcttt ggacatgccg gaggaggcca tcgagacttt gctgtgctac
2821 ctggagctgc acccacacca ctggctggag ctgctggcga ccacctatac ccattgccgt
2881 ctgaactgcc ctgggggccc tgcccagctc caggccctgg cccacaggtg tccccctttg
2941 gctgtgtgct tggcccagca gctgcctgag gacccagggc aaggcagcag ctccgtggag
3001 tttgacatgg tcaagctggt ggactccatg ggctgggagc tggcctctgt gcggcgggct
3061 ctctgccagc tgcagtggga ccacgagccc aggacaggtg tgcggcgtgg gacaggggtg
3121 cttgtggagt tcagtgagct ggccttccac cttcgcagcc cgggggacct gaccgctgag
3181 gagaaggacc agatatgtga cttcctctat ggccgtgtgc aggcccggga gcgccaggcc
3241 ctggcccgtc tgcgcagaac cttccaggcc tttcacagcg tagccttccc cagctgcggg
3301 ccctgcctgg agcagcagga tgaggagcgc agcaccaggc tcaaggacct gctcggccgc
3361 tactttgagg aagaggaagg gcaggagccg ggaggcatgg aggacgcaca gggccccgag
3421 ccagggcagg ccagactcca ggattgggag gaccaggtcc gctgcgacat ccgccagttc
3481 ctgtccctga ggccagagga gaagttctcc agcagggctg tggcccgcat cttccacggc
3541 atcggaagcc cctgctaccc ggcccaggtg tacgggcagg accgacgctt ctggagaaaa
3601 tacctgcacc tgagcttcca tgccctggtg ggcctggcca cggaagagct cctgcaggtg
3661 gcccgctgac tgcactgcat tgggggatgt cgggtagagc tggggttgtc agaggctagg
3721 gcagtgactg aggacctggg caaaacctgc cacagggtgt gggaacgagg aggctccaaa
3781 atgcagaata aaaaatgctc actttgtttt tatgggaaaa aaaaaaaaaa aaaaaaaaaa
〈210〉2
〈211〉 24
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg1
gcaagcgcggaggccgggcgggcg(SEQ ID NO:2)
〈210〉3
〈211〉 23
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg2
ggcgtgggagcgcgcgttccgac(SEQ ID NO:3)
〈210〉4
〈211〉 24
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg3
acgtggaggcggcgccggaggaga(SEQ ID NO:4)
〈210〉5
〈211〉 24
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg4
cagcggctcaaggccaatctgaaa(SEQ ID NO:5)
〈210〉6
〈211〉 22
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg5
ccaaggccaggccggctccagc(SEQ ID NO:6)
〈210〉7
〈211〉 24
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg6
cagtgaggtcccagattttctggg(SEQ ID NO:7)
〈210〉8
〈211〉 22
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg7
aacttctgatccctggtgagtc(SEQ ID NO:8)
〈210〉9
〈211〉 22
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg8
agtggaggcgagaagcggagat(SEQ ID NO:9)
〈210〉10
〈211〉 23
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg9
gagccctgggagagccccgcaca(SEQ ID NO:10)
〈210〉11
〈211〉 23
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg10
caagctagggctgggaaggctga(SEQ ID NO:11)
〈210〉12
〈211〉 25
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg11
ctactccctggggccctcagggcag(SEQ ID NO:12)
〈210〉13
〈211〉 24
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg12
gccggctgaggtgttccaggccct(SEQ ID NO:13)
〈210〉14
〈211〉 23
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg13
gtgcagtcatgcggatcctgtct(SEQ ID NO:14)
〈210〉15
〈211〉 22
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg14
actcatggatgaccaggtgtct(SEQ ID NO:15)
〈210〉16
〈211〉 21
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg15
caaggcggcctgcatacactc(SEQ ID NO:16)
〈210〉17
〈211〉 24
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg16
gcaaccggcgcgaggacacagagc(SEQ ID NO:17)
〈210〉18
〈211〉 23
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg17
ggcctttgggatggggctggacc(SEQ ID NO:18)
〈210〉19
〈211〉 24
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg18
gcggcggccgaagaggcgccagag(SEQ ID NO:19)
〈210〉20
〈211〉 22
〈212〉DNA
〈213〉人工序列
〈400〉RecQL4-sg19
gcaggcctgatctaggctcaga(SEQ ID NO:20)
〈210〉21
〈211〉 27
〈212〉DNA
〈213〉人工序列
〈400〉上游引物
ccggcaagcgcggaggccgggcgggcg（SEQ ID NO：21）
〈210〉22
〈211〉 27
〈212〉DNA
〈213〉人工序列
〈400〉下游引物
aaacgcccgcccggcctccgcgcttgc（SEQ ID NO：22）

Claims (4)

1.用于CRISPR-Cas9特异性敲除的人RecQL4基因，其序列如SEQ ID NO：1所示。
2.在CRISPR-Cas9特异性敲除人RecQL4基因中用于特异性靶向RecQL4基因的sgRNA，其特征在于，其对应的DNA序列如序列表SEQIDNO：2 -18任一所示。
3. 一种利用CRISPR-Cas9系统在体外特异性敲除人RecQL4基因的方法，其中使用的sgRNA如SEQ ID No：2-18任一所示。
4.根据权利要求2所述的sgRNA在制备用于治疗乳腺癌的药物中的用途。
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