CN105567738A - 使用基因组编辑技术CRISPR-Cas9诱导CCR5Δ32缺失的方法 - Google Patents
使用基因组编辑技术CRISPR-Cas9诱导CCR5Δ32缺失的方法 Download PDFInfo
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Abstract
本发明涉及一种新的基因组编辑技术CRISPR-Cas9成功诱导细胞趋化因子受体CCR5基因突变成CCR5Δ32缺失型基因的方法。CCR5是艾滋病毒(HIV)入侵人宿主细胞重要的辅助受体。CCR5Δ32缺失为CCR5编码区发生32个碱基的缺失，导致第185位氨基酸后的序列发生改变，并且提前终止。CCR5Δ32双等位基因纯合子缺失对艾滋病毒(HIV)感染具有天然抗性，不感染艾滋病毒。本发明同时使用了慢病毒包装系统和CRISPR技术，由于慢病毒感染宿主范围广的特性，所以该发明可应用于骨髓干细胞和CD4T细胞等细胞中，有望成为治疗艾滋病或者其他疾病的药物。
Description
技术领域
本发明属于生物医药领域，涉及一种新的基因组编辑技术CRISPR-Cas9诱导CCR5Δ32缺失的方法。
背景技术
由人类免疫缺陷病毒(HumanImmunodeficiencyVirus，HIV)或称艾滋病病毒引起的艾滋病(AcquiredImmuneDeficiencySyndrome，AIDS)在全球已经流行了30多年。艾滋病属于烈性传染病，HIV病毒可以直接攻击人体免疫系统，导致人体免疫机能缺陷，最终因各种机会性感染和肿瘤导致患者死亡。HIV主要分成2个型，HIV-1和HIV-2型。在全球引起艾滋病的主要是HIV-1，HIV-2仅限于非洲部分地区和其他地区的散在报道。据世界卫生组织(WorldHealthOrganization，WHO)报道，截止2013年底，全世界约有3500万HIV-1存活感染者，另有大约3000万人已经死亡，总计大约6500万人。2013年新发HIV-1感染者约为210万，当年死于艾滋病的病人约为150万(http://www.who.int)。迄今为止，依然没有有效的抗艾滋病疫苗。虽然抗逆转录病毒药物以及抗病毒疗法(antiretroviraltherapy，ART)已经取得很大的进展，能够有效控制病毒的复制，但依然不能彻底清除病人体内的HIV病毒，不能从根本上治愈艾滋病,并且由于抗逆转录疗法所需周期长,给病人家庭也带来了巨大的精神压力和经济压力。
我国也同样经历了艾滋病的打击。早在1989年，一例血友病患者因输血感染艾滋病。九十年代，云南吸毒人群艾滋病爆发流行。进入二十一世纪，我国艾滋病的流行逐渐从采供血人群、吸毒人群向性传播人群转变，包括同性恋和异性恋传播。据中国卫生部和联合国艾滋病规划署报道，截至2011年底，估计中国存活HIV-1感染者和艾滋病病人78万人(62～94万人)，女性占28.6％；艾滋病(AIDS)病人15.4万人(14.6～16.2万人)；全人群感染率为0.058％(0.046％～0.070％)。估计2011年当年新发HIV-1感染者4.8万人(4.1～5.4万人)，2011年艾滋病相关死亡2.8万人(2.5～3.1万人)。同样，中国的艾滋病流行情况也不容乐观。
HIV病毒主要攻击人的免疫系统，首先通过识别并结合细胞表面的CD4受体(CD是(ClusterofDifferentiation的缩写)是位于细胞膜表面一类分化抗原的总称，CD后的序号代表一个或一类分化抗原分子)。CD4是HIV感染所必须的，在后续过程中HIV病毒分子还需要与辅助受体相互作用，辅助受体主要为趋化因子受体CCR5(C-CChemokinereceptortype5)和CXCR4(C-X-Cchemokinereceptortype4)受体。HIV病毒成功结合受体蛋白和辅助受体蛋白后，导致病毒的包膜和细胞膜结构发生改变，从而发生融合，病毒进入细胞内部。CCR5是一个7次跨膜蛋白(如图1)，含有352个氨基酸，是细胞表面的一种正常的趋化因子受体，同时也是HIV进入靶细胞CD4阳性细胞的主要辅助受体。人们发现，在欧洲高加索人中存在着一些可以抵抗HIV病毒的人，测序表明，在这些人中正常的CCR5基因突变成了CCR5Δ32基因。CCR5Δ32缺失为CCR5基因编码区第794-825位的32个碱基的缺失(如图2)，使其185位后的氨基酸发生改变，导致翻译提前终止，产生了一种仅有215个氨基酸的缺失蛋白，这种缺失蛋白由于丧失了5、6、7三个跨膜区域，不能正常定位于细胞膜上，只能停留在内质网中。拥有这种突变的细胞由于缺少了能与病毒融合所需要的辅助受体，使其具有了能够抵抗HIV的能力。若细胞内的CCR5双等位基因都带有Δ32缺失，则该细胞为纯合子，记作CCR5Δ32/Δ32；若仅有一个Δ32缺失突变，另一个为野生型，则为杂合子，记作CCR5Δ32/WT。研究表明，CCR5Δ32/Δ32纯合子缺失对HIV具有天然抗性，不能感染HIV病毒；CCR5Δ32/WT杂合子缺失，可以延长艾滋病的发病期。
2009年，德国医生报道，柏林的一位HIV感染者叫TimothyBrown,在诊断为HIV感染10年，并接受抗病毒治疗4年以后，诊断出患有急性髓细胞白血病(acutemyeloidleukemia)。医生为他进行了异体干细胞移植，干细胞移植的供者是CCR5Δ32/Δ32纯合子。患者在停止抗病毒治疗20个月，接受了两次干细胞移植后，没有发现病毒的反弹。更重要的是，停药7年以后，依然没有发现病毒反弹。这是世界上第一个被认为是治愈的HIV感染者，或者称为功能性治愈，即停止抗病毒治疗后，没有病毒反弹。
调查发现，CCR5Δ32/Δ32缺失纯合子在欧洲高加索人群中稍高(4-10％)，但是在非洲和亚洲人群中很少存在，中国人中只有0－0.19％。在中国，由于供者的稀少，以及存在异体移植的排斥反应的风险，试图寻找CCR5Δ32/Δ32缺失的供者进行骨髓移植，复制柏林病人是不现实的。本发明试图使用新的方法完成这一任务。
HIV病毒基因组为核糖核酸RNA，因带有逆转录酶，分类上属于逆转录病毒科、慢病毒属。这一属的病毒致病，需要很长的潜伏期，缓慢发病，因此称为慢病毒。慢病毒载体是以HIV-1为基础发展起来的基因治疗载体，去除了HIV-1的致病基因，模拟病毒感染细胞的过程，它对分裂细胞和非分裂细胞均具有感染能力，适用于较难转染外源基因的细胞，如淋巴细胞、原代细胞、神经元细胞、干细胞等。该载体可以将外源基因有效地整合在宿主细胞的基因组中，从而达到长时间、稳定地表达外源基因。在实验过程中，需要将携带外源基因的目的质粒和包装质粒同时共转染到包装细胞中，以此来产生慢病毒颗粒，包装好的假病毒颗粒会分泌到细胞外的培养基中，离心取得上清液后，直接用于宿主细胞的感染，包装好的慢病毒颗粒感染宿主细胞后，目的质粒会在逆转录酶的作用下逆转录为cDNA，在整合酶的作用下随机整合到宿主细胞基因组中，使得目的基因在宿主细胞中可以稳定长期表达。
最近，一种新的基因组编辑技术──成簇的、规律间隔的短回文重复序列及其相关蛋白CRISPR-Cas技术[clusteredregularlyinterspacedshortpalindromicrepeat(CRISPR)andCRISPRassociatedprotein(Cas)]日益受到重视并得到广泛应用。CRISPR序列普遍存在于细菌和古细菌中，是在不断进化的过程中形成的一种细菌对抗细菌的病毒──噬菌体进攻的免疫防御系统。CRISPR位点通常由短的高度保守的重复序列(repeats)组成，重复序列之间由不同长度的间隔序列(spacer)所隔开，间隔序列一般被认为来源于噬菌体或外源DNA序列，类似于免疫记忆，当具有同样基因序列的外源DNA再次侵染细胞时，该间隔序列就可以识别并与外源序列结合，然后在一系列相关蛋白的作用下将外源序列进行切割，以此达到保护自身的目的。CRISPR系统根据参与防御过程的Cas蛋白的不同，可分为三种类型。其中II型CRISPR系统由于其简单性应用范围更加广泛，它仅需要一种Cas9蛋白就可以完成整个外源基因序列的切割过程。自然情况下II型CRISPR系统还需要crRNA(CRISPRRNA)和transcrRNA(trans-activatingcrRNA)来共同引导Cas9对外源基因序列的识别及切割，为了简化这一系统，目前较为普遍使用的是crRNA和transcrRNA两者的融合体──引导RNA(guideRNA，gRNA)来帮助Cas9蛋白识别目标序列。CRISPR-Cas9技术通过gRNA识别原型间隔序列毗邻基序(protospaceradjacentmotif,PAM)位点与目标序列结合，在核酸酶Cas9的作用下，将目标位点处的双链DNA进行切割，导致双链断裂，从而诱发细胞的自我修复过程，细胞可以通过两种方式进行修复──同源重组或非同源末端连接，通过这两种修复途径可以产生特异性的基因修饰或造成少数核苷酸残基的缺失插入突变，从而导致了目标序列的基因发生突变。
已有一些报道使用该技术破坏CCR5整个基因，但是如果破坏CCR5的整个基因，其潜在的危害未知。而CCR5Δ32缺失在人群中已经存在，突变者可以健康地存活，没有见到任何异常。因此，诱导CCR5Δ32缺失更有意义。
本发明利用慢病毒包装系统在淋巴细胞中成功表达CRISPR-Cas9，成功获得了基因型为CCR5Δ32/Δ32纯合的单克隆细胞，为人们在基因水平治疗艾滋病上提供了新的思路。
发明内容
本发明目的是解决人工诱导CCR5Δ32缺失的问题，提供一种使用基因组编辑技术CRISPR-Cas9诱导HIV病毒辅助受体CCR5Δ32/Δ32缺失的方法，从而为可能的治愈艾滋病提供一种药物。
本发明的技术方案
使用基因组编辑技术CRISPR-Cas9诱导CCR5Δ32缺失的方法，该方法使用了基因组编辑技术CRISPR-Cas9技术，成功诱导了CCR5Δ32缺失，该方法的具体内容包括：
第一、引导RNA(gRNA)的设计
为达到获得CCR5Δ32/Δ32纯合子细胞的目的，针对于CCR5Δ32两侧的目标序列设计一对gRNA，左侧的目标序列为gRNA对应的DNA序列，如SEQIDNo.1或者SEQIDNo.3-19中的任一个序列，右侧的目标序列为gRNA对应的DNA序列，如SEQIDNo.2或者SEQIDNo.20-36中的任一个序列；我们发现选取这两个目标序列后的Cas9核酸酶切割结果和天然CCR5Δ32的缺失完全一样(如图2，箭头所示为Cas9切割位点)。
所述的目标序列还可以是SEQIDNo1.和SEQIDNo.20-36的任意组合；SEQIDNo2.和SEQIDNo.20-36的任意组合；……SEQIDNo19.和SEQIDNo.20-36的任意组合；由于CRISPR-Cas9的容错性,个别碱基发生改变,也可以被识别，因此只要带有种子DNA序列“5’taatgtc3’”和“5’gactgta3’”的gRNA针对于CCR5Δ32缺失就在本发明保护范围内。
第二、根据第一步设计的gRNA，将对应的DNA插入到CRISPR-Cas9载体质粒中，构建功能质粒,该质粒具有以下特点：
①该质粒带有核酸酶Cas9表达读码框、CRISPR其他相关基因序列和慢病毒包装信号，质粒图谱如图3所示(质粒载体lenti-CRISPR-v2来自于美国麻省理工学院的张峰实验室)
②针对CCR5Δ32位点两端处的gRNA，gRNA序列如第一步所述；
研究表明，对于同一处目标序列来说，不仅仅只有野生型的Cas9核酸酶才能发生切割，对于一个目标序列来说，同时使用两个Cas9切口酶(Cas9的突变体)也可进行切割，所以使用Cas9切口酶或者Cas9的突变体也可达到本发明的实验目的，或者其他的核酸酶FokI。
第三、包裹CRISPR-Cas9质粒的慢病毒颗粒的制备：
将第二步构建的CRISPR-Cas9质粒以及和包装质粒PMD2.G、psPAX2共同转染到HEK293T细胞中，一段时间后收取细胞上清液，该上清中含有我们所需要的慢病毒颗粒。
第四、将第三步获得的慢病毒颗粒感染目的细胞，就能够获得CCR5Δ32/Δ32纯合缺失型细胞。
本发明的优点和积极效果：
(1)使用了一种新型的基因组编辑技术CRISPR-Cas9和慢病毒包装系统，成功诱导了CCR5Δ32缺失。
(2)诱导成功的CCR5Δ32/Δ32纯合缺失型细胞，可以作为一种新型药物治疗艾滋病。
总之，本发明同时使用了慢病毒包装系统和CRISPR-Cas9技术，成功获得CCR5Δ32/Δ32纯合缺失型淋巴细胞，并且由于慢病毒感染范围广，该发明也可应用于神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞、淋巴细胞等多种类型的细胞中。
附图说明：
图1是CCR5蛋白示意图，标注7个跨膜区。
图2是CCR5基因示意图,方框为CCR5Δ32缺失基因，箭头为Cas9切割位点。
图3是实验中所用的lenti-CRISPR-v2载体图谱,标注位点为限制性酶切位点。
图4是整个实验过程的流程图。
图5是显微镜图片，带有绿色荧光蛋白(GFP)的慢病毒载体感染人淋巴细胞系Jurkat的结果；A图是光学显微镜结果(放大倍数10×10)，B图为荧光显微镜结果(同样视野，放大倍数10×10)，浅白色为GFP阳性细胞，黑色为GFP阴性细胞。
图6是T7核酸内切酶(T7E1)酶切法检测gRNA在淋巴细胞Jurkat中CCR5基因的编辑效率。
图7是T7E1酶切法检测不同单克隆细胞组在CCR5基因处的切割情况。
图8是野生型细胞和获得的CCR5Δ32/Δ32单克隆细胞在CCR5Δ32位点处的测序图谱。
图9是获得的两个CCR5Δ32/Δ32单克隆细胞测序序列与野生型CCR5测序序列对比图。
具体实施方式
实施例一：携带CRISPR-Cas9系统的质粒重构及包裹重构质粒的慢病毒颗粒制备
1.gRNA的选择与设计
(1)根据实验目的我们需要设计一对gRNA同时进行编辑修饰CCR5基因，但是由于CCR5基因的限制性以及PAM位点的限制性，gRNA的选择是受限制的,左侧gRNA，如SEQIDNo.1，或者SEQNo.3-19。右侧序列，如SEQIDNo.2或者SEQIDNo.20-36。或者这两侧gRNA的任意组合。
(2)根据gRNA序列，加入酶切位点，合成DNA，分别连入BsmBI酶切后的lenti-CRISPR-v2载体中，获得重新构建的质粒，并测序验证序列的正确性。整个实验流程，如图4。
2.慢病毒颗粒的制备及感染效率的鉴定
(1)在10mm的盘子中铺板HEK293T细胞，细胞总数为6×106，第二天将步骤1获得的两种带有一对gRNA序列的lenti-CRISPR-v2质粒和慢病毒包装质粒PMD2.G、psPAX2按照浓度比例是1：1：1：1的体积共转染到细胞中，在37℃，含有5％CO2的恒温培养箱中培养16h，然后离心去除全部的培养基，加入新鲜的DMEM完全培养基(加入10％胎牛血清和青链霉素双抗)进行培养，24h后离心收集细胞上清液，该上清液中含有我们所需要的慢病毒颗粒。将上清液分装到1.5ml的离心管中，每管1ml，保存在-80℃冰箱中待用。同时，将能表达GFP蛋白的pwpxld质粒也包装入慢病毒颗粒中，以此观察慢病毒颗粒感染淋巴细胞的效率大小。
(2)感染前铺淋巴细胞于六孔板中，每孔的细胞总数为2×105，所用培养基为不含抗生素的全培养基，第二天将包裹pwpxld的慢病毒颗粒加入到细胞中，同时加入能增加病毒感染效率的polybrene，调整其浓度为8μg/ml，37℃，1600rpm离心1h，离心结束后去除含有病毒液的培养基，加入新的完全培养基，培养48h后观察细胞中的GFP表达情况。根据细胞中GFP表达情况可以得出慢病毒感染效率可达到80％以上(如图5,显微镜照片)。
实施例二：包裹有重构lenti-CRISPR-v2质粒的慢病毒颗粒感染淋巴细胞后的结果
1.感染前铺淋巴细胞于六孔板中，每孔的细胞总数为2×105，所用培养基为不含抗生素的全培养基，第二天，将实施例一中获得的包裹有gRNA的lenti-CRISPR-v2质粒的慢病毒颗粒和包裹空的lenti-CRISPR-v2质粒的慢病颗粒分别加到细胞培养基中，同时加入能增加病毒感染效率的polybrene，调整其浓度为8μg/ml，37℃，1600rpm离心1h，离心结束后去除含有病毒液的培养基，加入新的完全培养基，培养72h后离心收集细胞，将收集的细胞一半转入新的培养瓶中继续培养，另一半用来提取细胞总基因组DNA用于后续分析。
2.以提取的细胞基因组DNA为模板，用正向引物SEQIDNo.37和反向引物SEQIDNo.38PCR扩增含有CCR5Δ32位点的序列，然后用T7核酸内切酶(T7E1)酶切法验证CRISPR系统的编辑效率(T7endonucleaseI，T7E1酶，T7E1可以识别有错配的DNA，并对其进行切割)。实验结果如图6所示，从图中可以看出，转染一对实施例一中获得的lenti-CRISPR-v2质粒组发生了切割，而空载体组没有，说明这套系统是工作的。
3.由于CRISPR-Cas9系统切割目标序列DNA双链后会引发细胞自身修复途径，可能会在断裂处造成不同数量的碱基的插入或缺失，为了进一步纯化细胞的基因型，首先经计数后取出100个细胞稀释至体积为1000μl，混合均匀后，分别取10μl的混合液加入到96孔板中，每孔再加入200μl的完全培养基放于培养箱中进行培养。第二天，观察每孔中的细胞数，挑选并标记仅有一个细胞的孔进行继续培养。两周后，将由单个细胞长成的单克隆细胞转移至24孔板中继续培养，待细胞长满后转移到6孔板中继续培养。
4.收集上述六孔板中的单克隆细胞，一半用于继续培养，另一半用于提取细胞的基因组。同样使用上述中的引物扩增一段CCR5基因序列，然后使用T7核酸内切酶酶切初步鉴定在这些单克隆细胞中CCR5基因是否发生了突变。实验结果如图7所示，可以看出所用的单克隆细胞组都发生了切割，说明有突变。下一步，我们用测序证明是否发生了CCR5Δ32缺失。
5.挑选5个不同单克隆细胞用来进一步的分析，首先各取1μl单克隆细胞的PCR产物，连入TA克隆载体中，分别获得20μl的连接产物，然后取5μl的连接产物转化进大肠杆菌感受态细胞DH5ɑ中，涂板挑取单克隆进行测序鉴定，测序结果显示其中有3个单克隆细胞都为CCR5Δ32/Δ32纯合型突变，另外两个在CCR5Δ32处发生了不同数量的碱基插入或缺失。部分测序结果如图8所示，部分序列比对图9所示。
本发明是利用慢病毒颗粒包裹我们所设计的CRISPR-Cas9系统进行感染淋巴细胞，成功诱导了CCR5Δ32/Δ32纯合子缺失。由于慢病毒感染范围广，所以该系统同样也可用于感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞、淋巴细胞等多种类型的细胞。
Claims (5)
1.使用基因组编辑技术CRISPR-Cas9诱导CCR5Δ32缺失的方法，其特征在于使用了基因组编辑技术——成簇的、规律间隔的短回文重复序列及其相关蛋白CRISPR-Cas9技术，成功诱导了CCR5Δ32缺失，该方法的具体内容包括：
第一、引导RNA(gRNA)的设计
为达到获得CCR5Δ32/Δ32纯合子细胞的目的，针对于CCR5Δ32两侧的目标序列设计一对gRNA，左侧的目标序列为gRNA对应的DNA序列SEQIDNo.1或者SEQIDNo.3-19中的任一个序列，右侧的目标序列为gRNA对应的DNA序列SEQIDNo.2或者SEQIDNo.20-36中的任一个序列；
第二、根据第一步设计的gRNA，将对应的DNA插入到CRISPR-Cas9载体质粒中，构建功能质粒,该质粒具有以下特点：
①该质粒带有核酸酶Cas9表达读码框、CRISPR其他相关基因序列和慢病毒包装信号；
②针对CCR5Δ32位点两侧的gRNA，如第一步所述；
第三、包裹CRISPR-Cas9质粒的慢病毒颗粒的制备：
将第二步构建的CRISPR-Cas9质粒和包装质粒PMD2.G、psPAX2共同转染到HEK293T细胞中，一段时间后收取细胞上清液，该上清中含有我们所需要的慢病毒颗粒；
第四、将第三步获得的慢病毒颗粒感染目的细胞，就能够获得CCR5Δ32/Δ32纯合子细胞。
2.根据权利要求1所述的方法，其特征在于所述的目标序列还可以是SEQIDNo1.和SEQIDNo.20-36的任意组合；SEQIDNo2.和SEQIDNo.20-36的任意组合；……SEQIDNo19.和SEQIDNo.20-36的任意组合；由于CRISPR-Cas9的容错性,个别碱基发生改变,也可以被识别，因此只要带有种子DNA序列“5’taatgtc3’”和“5’gactgta3’”的gRNA针对于CCR5Δ32缺失就在本发明保护范围内。
3.根据权利要求1所述的方法，其特征在于所述切割使用的核酸酶还可以是Cas9的突变体——Cas9切口酶，或者其他的Cas9突变体，或者核酸酶FokI。
4.根据权利要求1所述的方法，其特征在于所述的质粒载体可以不是慢病毒载体，而是腺病毒载体；或者其他的带有CRISPR-Cas9的任意质粒载体。
5.根据权利要求1所述的方法，其特征在于所述的方法可以不是慢病毒感染，可以是质粒直接转染。
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Application publication date: 20160511 |