CN107236739A - CRISPR/SaCas9特异性敲除人CXCR4基因的方法 - Google Patents

CRISPR/SaCas9特异性敲除人CXCR4基因的方法 Download PDF

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CN107236739A
CN107236739A CN201710444953.0A CN201710444953A CN107236739A CN 107236739 A CN107236739 A CN 107236739A CN 201710444953 A CN201710444953 A CN 201710444953A CN 107236739 A CN107236739 A CN 107236739A
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sequence
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杨通
刘佩
欧恩智
陈珺
朱智
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Shanghai Jieyi Biotechnology Co ltd
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Abstract

本发明公开了一种用于特异性靶向人CXCR4基因的sgRNA，其靶序列如SEQ ID NO:1～4中任一条序列所示。本发明还公开了包含有上述sgRNA的金黄色葡萄球菌CRISPR/SaCas9系统，以及用该系统特异性敲除人CXCR4基因的方法。本发明通过将特异性靶向人CXCR4基因的sgRNA与AAV‑CRISPR/SaCas9质粒连接成载体，包装成AAV感染细胞，简便、高效、特异地实现了CXCR4基因的敲除，从而有效解决了使用SpCas9靶向CXCR4进行艾滋病治疗的局限性。

Description

CRISPR/SaCas9特异性敲除人CXCR4基因的方法
技术领域
本发明涉及基因工程领域，特别是涉及基于金黄色葡萄球菌CRISPR/Cas9(CRISPR/SaCas9)系统特异性敲除人CXCR4基因的方法，以及用于特异性靶向人CXCR4基因的sgRNA。
背景技术
人类免疫缺陷病毒(human immunodeficiency virus，HIV)归属于逆转录病毒科慢病毒属中的人类慢病毒组，是一种感染人类免疫系统细胞的慢病毒，属反转录病毒的一种，又称为艾滋病毒，其感染引起的获得性免疫缺陷综合征(acquired immunodeficiencysyndrome，AIDS)又称艾滋病，是一种危害性极大的传染病。自发现的三十多年来，AIDS已累计导致两千余万人死亡，一直是一个巨大的公众健康难题，影响着全球3,530万人的生活。
HIV可分为HIV-1和HIV-2两种亚型，HIV-1的致病力强，是引起艾滋病的主要病原体。CD4+T细胞是HIV-1病毒感染过程的首要靶点。随后研究又发现仅有CD4分子并不能介导HIV-1病毒的侵入，同时还需要一种或几种辅助受体(coreceptor)。1996年证实，趋化因子受体CXCR4和CCR5是HIV-1病毒感染的辅助受体。T细胞向性X4HIV-1使用CD4和CXCR4进入细胞，而巨噬细胞向性R5HIV-1使用CD4和CCR5。双向性的毒株能使用CXCR4和CCR5作为共受体。其他趋化因子受体中的CCR3、CCR2、CCR8、CXCR6、CXCR7、CX3CR1能作为更限制亚群的HIV株的共受体发挥作用。
目前对艾滋病的治疗手段主要是控制HIV-1病毒的感染以及阻碍AIDS的发展，称为高效抗逆转录病毒治疗法(highly active antiretroviral therapy，HAART)，包括一系列抑制病毒各个繁殖阶段的化合物。HAART是能很大程度上减少细胞内病毒的繁殖和血浆病毒血症的发生，但对于新的宿主细胞不被HIV-1感染没有作用。为了从源头上预防健康的细胞被HIV-1感染，新的治疗方案需要彻底地破坏病毒的入侵途径，因而以CCR5和CXCR4为靶点的HIV-1受体拮抗剂越来越受关注，主要有趋化因子衍生物、非肽类小分子化合物、单克隆抗体、肽类化合物等。如SDF-1(CXCR4的天然配体)以及CCR5的CCL3、CCL4、CCL4-L1和CCL5配体能够抑制不同株HIV-1引起的细胞融合和感染。
尽管辅助受体拮抗剂可有效预防HIV-1感染，遏制艾滋病的流行，但是此类抑制剂的发展也面临着挑战。长期使用一种抑制剂，最终会使HIV-1产生耐药性。随着耐药性的不断出现，使得现有的抗HIV-1药物难以达到理想的治疗效果。因此，从基因组层面失活CCR5或CXCR4具有重要的治疗价值。
2008年，Sangamo成功的利用Engineered zinc finger nucleases(ZFNs)在CD4+T细胞上实现CCR5的敲除。如今这项名为SB-728-T的项目已被尝试应用于HIV感染人群的基因治疗，并已进入临床二期阶段。这一基因编辑产品通过敲除分离到病人的CD4+T细胞的CCR5，然后自体回输到病人，可以用于HIV感染的治疗。但如前面所提及的CCR5受体是巨噬细胞向性R5HIV-1的主要共受体，而T细胞向性X4HIV-1的主要共受体是CXCR4，敲除CCR5并不能治疗被双向性HIV-1感染的患者或者被T细胞向性X4HIV-1感染的患者。因此，通过基因层面敲除CXCR4在X4-向性和双向性HIV感染的患者中扩展此类治疗有明确的医学需求。
规律成簇间隔短回文重复系统(clustered regularly interspaced shortpalindromic repeat，CRISPR-associated，CRISPR/Cas9)是一种具有核酸内切酶活性的复合体，是细菌和古细菌在进化过程中形成的一种免疫防御体系，用以对抗外来病毒和外源DNA入侵。CRISPR/Cas9系统通过整合外源DNA片段到CRISPR中，利用CRISPR RNA(CRISPR-derived RNA，crRNA)和反式作用RNA(trans-activating RNA，tracrRNA)特异性地识别靶向序列，并对序列靶位点进行切割。在没有模板的条件下，发生非同源重组末端连接(Non-homologous end joining，NHEJ)，NHEJ修复的过程中往往会产生DNA的插入或缺失(Indel)，造成移码突变，导致基因敲除。或者是在有同源模板的情况下，可通过另一条同源重组(homologous recombination，HR)的修复途径进行修复，可实现对靶向基因的精确编辑，如引入特异突变或定点转基因。现在已经把两种小RNA(crRNA和tracrRNA)融合成一条RNA链，简称sgRNA(single guide RNA)，因此，能够设计、制备出精确性和特异性靶向目标基因的sgRNA也成为CRISPR/Cas9基因敲除的关键。
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。研究人员优先选择改造了来自化脓链球菌的Cas9酶(Streptococcus pyogenes Cas9,SpCas9)来改变高等生物的DNA，并已成为一系列高度通用的基因组修饰技术的基础。最近的一些研究利用CRISPR/SpCas9系统在CD4+T细胞或者CD34+干细胞对艾滋病毒的辅助受体CCR5进行基因修饰，在一定的程度上可以抑制HIV-1病毒的侵入，但是也存在着一定的脱靶率。
最近发现了一种新的CRISPR/Cas9系统，它是一种是来自金黄色葡糖球菌的Cas9酶(Staphylococcus aureus Cas9,SaCas9)，其基因敲除效率与之前的CRISPR/SpCas9系统相当，但是其大小却减少了25％。腺相关病毒(AAV)是基因治疗的最佳载体，但是由于其负载能力有限，使其包装SpCas9酶和其他必须的基因元件进入单个病毒颗粒存在一定的难度，而SaCas9的出现为这一问题的解决带来了新的希望。
SaCas9与SpCas9存在着不同之处，SaCas9主要识别5’-NNGRRT-3’特征区域的NGGRRT位点，然后对与sgRNA互补的DNA序列进行剪切，被剪切后断裂的DNA双链通过非同源末端直接连接(non-homologous end joining，NHEJ)或者同源重组(homology-directedrepair，HDR)的修补方法进行修复，修复后的DNA会由于某些碱基的随机插入、缺失、突变等使基因的序列发生改变从而抑制了基因的表达，由此在DNA水平上对基因进行定向敲除。而SpCas9的识别位点为PAM序列5’-NGG-3’，然后再对靶位点进行剪切。
使用SpCas9靶向CXCR4进行艾滋病治疗，存在以下问题：1)SpCas9基因较大，不能方便地利用AAV载体进行基因治疗；2)SpCas9在靶向精确性上有所不足。
SaCas9与SpCas9相比具有很多优点：1)SaCas9在哺乳动物体内具有很高的剪切效率，其所带有的sgRNA的长度范围为21-23个碱基，其本身的大小比SpCas9小25％，更有利于AAV载体携带其它相关基因进入单个病毒颗粒，经验证其与SpCas9可以达到相当的效率。2)在SaCas9系统中，把其sgRNA的第一个碱基替换成鸟嘌呤，使得SaCas9酶的活性大大提高，从而提高了其在机体内的剪切效率。3)SaCas9在靶向精确性上效率更高。
发明内容
本发明要解决的技术问题之一是提供一种用于特异性靶向人CXCR4基因的sgRNA。
为解决上述技术问题，本发明的用于特异性靶向人CXCR4基因的sgRNA，其靶序列为SEQ ID NO:1～4所示序列中的任意一条序列。
所述sgRNA的靶序列优选为SEQ ID NO:3或SEQ ID NO:4所示的序列。
本发明要解决的技术问题之二是提供一种CRISPR/SaCas9特异性敲除人CXCR4基因的方法，该方法用于非诊断和治疗目的，步骤包括：
1)在所述sgRNA的靶序列的5’端加上用于形成粘性末端的序列，得到正向寡核苷酸；在所述sgRNA的靶序列的互补链的两端加上用于形成粘性末端的序列，得到反向寡核苷酸；合成所述正向寡核苷酸和反向寡核苷酸，变性、退火，形成带有粘性末端的双链sgRNA寡聚核苷酸；
2)线性化腺相关病毒载体AAV-CRISPR/SaCas9，将所述双链sgRNA寡聚核苷酸连入线性化的AAV-CRISPR/SaCas9载体，转化感受态细胞，筛选获得阳性克隆，摇菌，抽提质粒；
3)将步骤2)提取的含有所述双链sgRNA寡聚核苷酸的AAV-CRISPR/SaCas9载体包装成腺相关病毒；
4)用步骤3)包装的腺相关病毒感染目的细胞，培养，收集被感染的目的细胞，进行酶切检测和克隆测序，确定CXCR4基因的敲除效率，并获得基因敲除的细胞。
本发明要解决的技术问题之三是提供一种特异性敲除人CXCR4基因的CRISPR/SaCas9系统，该系统包含有上述特异性靶向人CXCR4基因的sgRNA。
本发明通过将特异性靶向人CXCR4基因的sgRNA与AAV-CRISPR/SaCas9质粒连接成载体，包装成AAV感染细胞，实现了CXCR4基因的敲除，相比现有使用SpCas9靶向CXCR4进行艾滋病治疗的方法，本发明的方法不仅简便、高效，而且特异性高。
附图说明
图1为PX601质粒的结构示意图。
图2是T7E1酶切鉴定CRISPR/SaCas9介导的人CXCR4基因特异性切割。
图3是CRISPR/SaCas9介导的基因位点特异性人CXCR4基因切割测序结果。其中，箭头表示Cas9切割位点，下划线序列为PAM序列，(-)表示敲除。
具体实施方式
为对本发明的技术内容、特点与功效有更具体的了解，现结合附图和具体实施例，对本发明的技术方案做进一步详细的说明。
1.特异性靶向人CXCR4基因的sgRNA的设计
以HIV-1的辅助受体蛋白即宿主蛋白CXCR4(NCBI基因号：NM_001008540.2)作为靶标，依据SaCas9编辑规则(F.Ann Ran et al.2015)，在CXCR4基因上选择符合5’-(20N)NNGRRT-3’的序列。
sgRNA在CXCR4基因上的靶向位点位于不同剪切形式的共有外显子上的编码区(靶向位点位于基因外显子上的编码区，更容易引起基因的移码框突变，达到基因完全失活的目的)，避开CpG岛的区域。
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，以减少潜在的脱靶位点。
最后设计出4个靶向人CXCR4基因的sgRNA，序列分别为：
GATGATTTCCAGGAGGATGA(SEQ ID NO:1)
GGAAATCATCAAGCAAGGGT(SEQ ID NO:2)
GGAATAGTCAGCAGGAGGGC(SEQ ID NO:3)
GACAGGATGACAATACCAGG(SEQ ID NO:4)
2.特异性靶向人CXCR4基因的sgRNA的选择
根据以下两个原则：
1)靶向CXCR4基因的sgRNA的靶序列在CXCR4基因上不能离ATG起始子太近，以避免转录会合下游另一个ATG开始，而出现一个被截短的基因形式，导致不能保证基因完全失活。
2)sgRNA在CXCR4基因上的靶向位点位于整个基因的前中段部分。
从上述4个靶向人CXCR4基因的sgRNA中挑选出2个sgRNA(序列如SEQ ID NO:3～4所示)进行后续实验。
3.特异性靶向人CXCR4基因的sgRNA寡聚核苷酸的合成和构建
分别在上述挑选出的2个sgRNA(序列如SEQ ID NO:3～4所示)的5’端加上CACCG得到正向寡核苷酸(Forward oligo)。
分别在上述挑选出的2个sgRNA(序列如SEQ ID NO:3～4所示)的互补链的5’端加上AAAC，3’端加上C，得到反向寡核苷酸(Reverse oligo)。
序列分别如下所示：
正向寡核苷酸：5’-CACCGGGAATAGTCAGCAGGAGGGC-3’(SEQ ID NO:5)
反向寡核苷酸：5’-AAACGCCCTCCTGCTGACTATTCCC-3’(SEQ ID NO:6)
正向寡核苷酸：5’-CACCGGACAGGATGACAATACCAGG-3’(SEQ ID NO:7)
反向寡核苷酸：5’-AAACCCTGGTATTGTCATCCTGTCC-3’(SEQ ID NO:8)
分别合成上述正向寡核苷酸和反向寡核苷酸，然后成对变性、退火，形成带有BsaI酶切位点、可以连入腺相关病毒载体AAV-CRISPR/SaCas9(PX601，Addgene公司)的粘性末端小片段(即双链sgRNA寡聚核苷酸)。
变性、退火体系为：100μM正向寡核苷酸2.5μl，100μM反向寡核苷酸2.5μl，2×NEB缓冲液1μl，灭菌水4μl。
变性、退火的PCR程序为：95℃，5min；95～85℃，-2℃/s；85～25℃，-0.1℃/s；4℃保持。
4.利用CRISPR/SaCas9特异性敲除人CXCR4基因
1)用BsaI核酸内切酶切割AAV-CRISPR/SaCas9，使AAV-CRISPR/SaCas9质粒线性化(序列如SEQ ID NO:9所示)。
酶切体系为：400ng/μl PX601 2μg，1μl CutSmart缓冲液，1μl BsaI核酸内切酶(NEB，R0535L)，补水至50μl。
酶切条件为：37℃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。
酶切完成后，用AxyPrep PCR Clean up Kit(AP-PCR-250)纯化试剂盒纯化回收至20～40μl灭菌水中。
2)将上述变性、退火之后获得的可以连入PX601载体的双链sgRNA寡聚核苷酸与线性化的PX601质粒相连，分别获得PX601-sghCXCR4#3质粒和PX601-sghCXCR4#4质粒。
连接体系和条件为：50μM双链sgRNA寡聚核苷酸3μl，25ng/μl线性化的PX601质粒1μl，T4连接缓冲液1μl，T4连接酶(NEB，M0202S)0.5μl，灭菌水4.5μl。16℃孵育1小时。
3)将上述获得的连接产物转化DH5α感受态细胞(TransGen，CD201)，并涂Amp+平板(50μg/ml)，挑取克隆。用U6通用引物，经常规测序方法鉴定获得阳性克隆。
U6通用引物为：GGACTATCATATGCTTACCG(SEQ ID NO:10)
4)37℃摇床摇菌过夜，培养阳性克隆，并用AxyPrep Plasmid Miniprep Kit(AP-MN-P-250)试剂盒抽提质粒，获得PX601-sghCXCR4#3质粒(序列如SEQ ID NO:11所示)和PX601-sghCXCR4#4质粒(序列如SEQ ID NO:12所示)。
5.包装含有sgRNA的腺相关病毒获得CXCR4基因敲除细胞
按照AAV Helper-Free System(Aglilent Technologies,#240071)，将上述设计构建的含有识别人CXCR4基因的sgRNA的AAV-CRISPR/SaCas9载体包装成腺相关病毒。具体过程如下：
1)转染前24小时铺AAV-293细胞(购自CCTCC)于一个直径10cm的细胞培养皿中；
2)待细胞密度细胞达到80～90％时，以标准的Lipofectamine3000转染，将上述质粒共转染细胞，转染后6～8小时换液。
转染体系为：2X Opti-MEM 500μl，1μg/μl pAAV-RC质粒10μl(购于AgilentTechnologies)，1μg/μl pHelper质粒10μl(购于Agilent Technologies)，1μg/μl PX601-sghCXCR4#3或PX601-sghCXCR4#4质粒10μl，Lipid reagent(脂质体试剂)30μl。
在500μl Opti-MEM中加入三种不同量的质粒DNA，同样用500μl Opti-MEM稀释Lipid reagent，将稀释后的DNA按1:1的比例加入到转染试剂中，混匀静止放置20min后，加入到培养皿里，培养7小时后换液。
3)转染后72小时收集病毒(细胞上清和细胞裂解液)过滤，然后对病毒上清进行浓缩，在4℃，70000rpm，超速离心2小时，弃去上清后加入一定量的培养基重悬，分装后-80℃保存。
4)病毒滴度检测：将病毒上清以10倍梯度稀释，利用病毒计数仪进行病毒滴度的初步测定。将病毒分别感染293T细胞，48小时之后药物检测阳性细胞比率，并以比率在0.1～10％之间的稀释度进一步精确计算出病毒滴度。
6.利用CRISPR/SaCas9重组腺相关病毒特异敲除人CXCR4基因
用上述包装的腺相关病毒以MOI为100(1ml病毒上清加到含4mlDMEM细胞中)感染HEK-293T细胞，72小时后收集细胞，做T7E1酶切检测和TA克隆测序，检测CXCR4基因的敲除效率，以确认CXCR4基因已敲除。
T7E1酶切检测方法如下：
1)将收集的细胞在裂解液(10μM Tris-HCl，0.4M NaCl，2μM 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50μl去离子水中。
2)以步骤1)提取的HEK293T细胞基因组为模板，使用hCXCR4-test-F和hhCXCR4-test-R为引物进行PCR扩增，得到444bpPCR产物，用AxyPrep PCR clean up kit试剂盒纯化获得PCR回收产物，取200ng统一稀释到20μl，进行变性、退火，程序为：95℃，5min；95～85℃，-2℃/s；85～25℃，-0.1℃/s；4℃保持。
hCXCR4-test-F：ACAGTCAACCTCTACAGCAG(SEQ ID NO:13)
hhCXCR4-test-R：GAAGGAGTCGATGCTGATCC(SEQ ID NO:14)
3)在20μl体系中加入T7E1 0.3μl，37℃酶切30分钟，然后加入2μl 10X LoadingBuffer，用2.5％的琼脂糖胶电泳检测。
发生断裂末端连接修复的基因组由于与原基因组不完全匹配，会被T7E1切割，在琼脂糖胶电泳图上显示出较小的条带，如图2所示，加入针对人CXCR4的sgRNA的样品都出现了切割条带，而且具有很高的效率。
TA克隆测序方法如下：
1)将T7E1酶切检测步骤2)获得的PCR回收产物用rTaq进行加A反应。
加A反应体系为：700～800ng PCR，回收产物，10X缓冲液(无Mg2+)5μl，Mg2+3μl，dNTP4μl，0.5μl rTaq(TAKARA，R001AM)，补水至50μl。
加A反应条件为：37℃温育30分钟，取1μl产物与pMD19-T vector(TAKARA，3271)连接并转化DH5α感受态细胞(TransGen，CD201)。
2)挑取单克隆，使用hCXCR4-test-F引物(SEQ ID NO:13)测序。
测序结果发现：靶基因CXCR4在编码区缺失了2个碱基或8个碱基，形成移码框突变，提前出现终止密码子，基因敲除成功(如图3所示)。
<110> 上海捷易生物科技有限公司
<120> CRISPR/SaCas9特异性敲除人CXCR4基因的方法
<130> Jeayea20160601
<160> 14
<170> PatentIn version 3.3
<210> 1
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<212> DNA
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<220>
<221> misc_feature
<223>
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ggaaatcatc aagcaagggt 20
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<221> misc_feature
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ggaatagtca gcaggagggc 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
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gacaggatga caataccagg 20
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caccgggaat agtcagcagg agggc 25
<210> 6
<211> 25
<212> DNA
<213> 人工序列
<220>
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aaacgccctc ctgctgacta ttccc 25
<210> 7
<211> 25
<212> DNA
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<221> misc_feature
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caccggacag gatgacaata ccagg 25
<210> 8
<211> 25
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<400> 8
aaaccctggt attgtcatcc tgtcc 25
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<213> 人工序列
<220>
<221> misc_feature
<223> 质粒
<400> 9
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180
agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240
ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300
tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360
atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420
ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480
gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540
ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600
tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660
aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720
tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc 780
ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa ctacatcctg ggcctggaca 840
tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg 900
ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga 960
gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc 1020
tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg 1080
ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc 1140
acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg 1200
agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg 1260
ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat 1320
tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc 1380
accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct 1440
actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg 1500
agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct 1560
acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg 1620
agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga 1680
agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta 1740
agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg 1800
acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga 1860
ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc 1920
tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg 1980
gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca 2040
acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt 2100
cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga 2160
agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc 2220
ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga 2280
tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga 2340
ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg 2400
aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct 2460
tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca 2520
acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt 2580
acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc 2640
tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg 2700
acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat 2760
acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg 2820
tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta 2880
agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg 2940
ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc 3000
agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca 3060
aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt 3120
acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca 3180
cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca 3240
aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc 3300
accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga 3360
agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa 3420
aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc 3480
tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc 3540
cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc 3600
tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat gaggaagcta 3660
agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac aacaacgatc 3720
tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc 3780
ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac atgaacgaca 3840
agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt aagaagtaca 3900
gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct cagatcatca 3960
aaaagggcaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat 4020
cctacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgcttacc 4080
catacgatgt tccagattac gcttaagaat tcctagagct cgctgatcag cctcgactgt 4140
gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 4200
aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 4260
taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 4320
agagaatagc aggcatgctg gggaggtacc tgagggccta tttcccatga ttccttcata 4380
tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac 4440
aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt 4500
tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga 4560
tttcttggct ttatatatct tgtggaaagg acgaaacacc ggagaccacg gcaggtctca 4620
gttttagtac tctggaaaca gaatctacta aaacaaggca aaatgccgtg tttatctcgt 4680
caacttgttg gcgagatttt tgcggccgca ggaaccccta gtgatggagt tggccactcc 4740
ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 4800
ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat 4860
gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca 4920
tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 4980
accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 5040
gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 5100
tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 5160
gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 5220
agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat 5280
ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 5340
tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca 5400
atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 5460
ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 5520
agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 5580
gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 5640
ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 5700
aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 5760
aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 5820
cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 5880
ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 5940
cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 6000
ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 6060
gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 6120
gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 6180
acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6240
cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 6300
acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 6360
ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 6420
ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 6480
ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 6540
atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 6600
ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 6660
attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 6720
ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 6780
aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 6840
aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 6900
ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 6960
tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 7020
ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 7080
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 7140
agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 7200
gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7260
ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 7320
tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 7380
tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 7440
cacatgt 7447
<210> 10
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<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 10
ggactatcat atgcttaccg 20
<210> 11
<211> 7448
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 质粒
<400> 11
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180
agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240
ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300
tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360
atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420
ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480
gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540
ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600
tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660
aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720
tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc 780
ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa ctacatcctg ggcctggaca 840
tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg 900
ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga 960
gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc 1020
tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg 1080
ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc 1140
acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg 1200
agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg 1260
ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat 1320
tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc 1380
accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct 1440
actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg 1500
agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct 1560
acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg 1620
agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga 1680
agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta 1740
agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg 1800
acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga 1860
ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc 1920
tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg 1980
gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca 2040
acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt 2100
cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga 2160
agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc 2220
ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga 2280
tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga 2340
ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg 2400
aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct 2460
tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca 2520
acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt 2580
acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc 2640
tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg 2700
acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat 2760
acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg 2820
tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta 2880
agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg 2940
ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc 3000
agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca 3060
aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt 3120
acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca 3180
cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca 3240
aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc 3300
accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga 3360
agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa 3420
aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc 3480
tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc 3540
cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc 3600
tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat gaggaagcta 3660
agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac aacaacgatc 3720
tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc 3780
ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac atgaacgaca 3840
agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt aagaagtaca 3900
gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct cagatcatca 3960
aaaagggcaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat 4020
cctacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgcttacc 4080
catacgatgt tccagattac gcttaagaat tcctagagct cgctgatcag cctcgactgt 4140
gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 4200
aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 4260
taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 4320
agagaatagc aggcatgctg gggaggtacc tgagggccta tttcccatga ttccttcata 4380
tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac 4440
aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt 4500
tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga 4560
tttcttggct ttatatatct tgtggaaagg acgaaacacc gggaatagtc agcaggaggg 4620
cgttttagta ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg 4680
tcaacttgtt ggcgagattt ttgcggccgc aggaacccct agtgatggag ttggccactc 4740
cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 4800
gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga 4860
tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc 4920
atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 4980
gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 5040
cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 5100
atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag 5160
tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 5220
tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga 5280
tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 5340
atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac 5400
aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 5460
gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 5520
gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct 5580
cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg 5640
tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 5700
aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 5760
gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 5820
ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 5880
gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 5940
tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 6000
attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 6060
tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 6120
agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 6180
aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 6240
tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 6300
cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 6360
tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 6420
tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 6480
tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 6540
tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 6600
aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 6660
gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 6720
tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 6780
aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 6840
aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 6900
tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 6960
gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 7020
cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 7080
acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 7140
cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 7200
cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 7260
aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 7320
gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 7380
atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 7440
tcacatgt 7448
<210> 12
<211> 7448
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 质粒
<400> 12
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180
agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240
ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300
tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360
atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420
ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480
gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540
ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600
tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660
aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720
tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc 780
ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa ctacatcctg ggcctggaca 840
tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg 900
ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga 960
gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc 1020
tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg 1080
ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc 1140
acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg 1200
agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg 1260
ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat 1320
tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc 1380
accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct 1440
actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg 1500
agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct 1560
acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg 1620
agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga 1680
agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta 1740
agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg 1800
acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga 1860
ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc 1920
tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg 1980
gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca 2040
acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt 2100
cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga 2160
agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc 2220
ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga 2280
tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga 2340
ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg 2400
aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct 2460
tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca 2520
acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt 2580
acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc 2640
tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg 2700
acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat 2760
acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg 2820
tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta 2880
agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg 2940
ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc 3000
agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca 3060
aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt 3120
acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca 3180
cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca 3240
aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc 3300
accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga 3360
agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa 3420
aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc 3480
tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc 3540
cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc 3600
tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat gaggaagcta 3660
agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac aacaacgatc 3720
tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc 3780
ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac atgaacgaca 3840
agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt aagaagtaca 3900
gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct cagatcatca 3960
aaaagggcaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat 4020
cctacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgcttacc 4080
catacgatgt tccagattac gcttaagaat tcctagagct cgctgatcag cctcgactgt 4140
gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 4200
aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 4260
taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 4320
agagaatagc aggcatgctg gggaggtacc tgagggccta tttcccatga ttccttcata 4380
tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac 4440
aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt 4500
tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga 4560
tttcttggct ttatatatct tgtggaaagg acgaaacacc ggacaggatg acaataccag 4620
ggttttagta ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg 4680
tcaacttgtt ggcgagattt ttgcggccgc aggaacccct agtgatggag ttggccactc 4740
cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 4800
gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga 4860
tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc 4920
atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 4980
gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 5040
cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 5100
atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag 5160
tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 5220
tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga 5280
tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 5340
atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac 5400
aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 5460
gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 5520
gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct 5580
cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg 5640
tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 5700
aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 5760
gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 5820
ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 5880
gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 5940
tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 6000
attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 6060
tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 6120
agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 6180
aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 6240
tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 6300
cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 6360
tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 6420
tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 6480
tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 6540
tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 6600
aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 6660
gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 6720
tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 6780
aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 6840
aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 6900
tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 6960
gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 7020
cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 7080
acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 7140
cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 7200
cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 7260
aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 7320
gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 7380
atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 7440
tcacatgt 7448
<210> 13
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 13
acagtcaacc tctacagcag 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 14
gaaggagtcg atgctgatcc 20

Claims (10)

1.用于特异性靶向人CXCR4基因的sgRNA，其特征在于，所述sgRNA的靶序列为SEQ IDNO:1～4所示序列中的任意一条序列。
2.根据权利要求1所述的用于特异性靶向人CXCR4基因的sgRNA，其特征在于，所述sgRNA的靶序列如SEQ ID NO:3或SEQ ID NO:4所示。
3.CRISPR/SaCas9特异性敲除人CXCR4基因的方法，该方法用于非诊断和治疗目的，其特征在于，步骤包括：
1)在权利要求1或2所述的sgRNA的靶序列的5’端加上用于形成粘性末端的序列，得到正向寡核苷酸；在权利要求1或2所述的sgRNA的靶序列的互补链的两端加上用于形成粘性末端的序列，得到反向寡核苷酸；合成所述正向寡核苷酸和反向寡核苷酸，变性、退火，形成带有粘性末端的双链sgRNA寡聚核苷酸；
2)线性化腺相关病毒载体AAV-CRISPR/SaCas9，将所述双链sgRNA寡聚核苷酸连入线性化的AAV-CRISPR/SaCas9载体，转化感受态细胞，筛选获得阳性克隆，摇菌，抽提质粒；
3)将步骤2)提取的含有所述双链sgRNA寡聚核苷酸的AAV-CRISPR/SaCas9载体包装成腺相关病毒；
4)用步骤3)包装的腺相关病毒感染目的细胞，培养，收集被感染的目的细胞，进行酶切检测和克隆测序，确定CXCR4基因的敲除效率，并获得基因敲除的细胞。
4.根据权利要求3所述的方法，其特征在于，步骤1)，在所述靶序列的5’端加上CACCG得到正向寡核苷酸；在所述靶序列的互补链的5’端加上AAAC，3’端加上C，得到反向寡核苷酸。
5.根据权利要求4所述的方法，其特征在于，所述正向寡核苷酸的序列如SEQ ID NO:5所示，所述反向寡核苷酸的序列如SEQ ID NO:6所示。
6.根据权利要求4所述的方法，其特征在于，所述正向寡核苷酸的序列如SEQ ID NO:7所示，所述反向寡核苷酸的序列SEQ ID NO:8所示。
7.根据权利要求3所述的方法，其特征在于，步骤1)，所述双链sgRNA寡聚核苷酸带有BsaI酶切位点；步骤2)，用BsaI核酸内切酶切割AAV-CRISPR/SaCas9载体，使所述载体线性化。
8.根据权利要求3所述的方法，其特征在于，步骤2)，所述感受态细胞为DH5α感受态细胞；步骤4)，所述目的细胞为HEK-293T细胞，所述酶切检测为T7E1酶切检测，所述克隆测序为TA克隆测序。
9.根据权利要求8所述的方法，其特征在于，步骤4)，T7E1酶切检测中使用的PCR扩增引物对的序列如SEQ ID NO:13～14所示。
10.特异性敲除人CXCR4基因的CRISPR/SaCas9系统，其特征在于，所述CRISPR/SaCas9系统包含有权利要求1或2所述的特异性靶向人CXCR4基因的sgRNA。
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