CN105331627A - 一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法 - Google Patents
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Abstract
本发明一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法。在对含有内源CRISPR-Cas系统的原核生物进行基因组编辑时，只需构建一个同时携带人工CRISPR簇和供体DNA的编辑质粒，在内源CRISPR系统对基因组发生DNA干涉后通过同源重组达到对基因组的编辑。其最大的优点在于：应用宿主范围广，所有含有内源CRISPR-Cas系统的细菌和古菌均可操作；可用于多种编辑方式，缺失、插入和点突变等均可操作；更高的编辑效率，筛选阳性率高，背景低；流程简单，时间周期短，大大减轻原核生物基因组编辑的工作量。
Description
技术领域
本发明属于基因组学及基因工程和生物技术领域，具体涉及到一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法。
背景技术
CRISPR-Cas系统作为一种原核生物抵御病毒等外源入侵核酸的获得性免疫系统，广泛存在于大约90％的古菌和40％细菌中(VanderOostJetal.,2014；BarrangouRetal.,2014)。CRISPR-Cas系统被划分为三个主要类型：I型，II型和III型；它们分别有一个标志性蛋白：Cas3，Cas9和Cas10(MakarovaKSetal.,2011)。II型CRISPR系统仅需要Cas9一个蛋白与一条crRNA和trans-actingRNA行使DNA干涉活性(DeltchevaEetal.,2011；GasiunasGetal.,2012)。简单的II型CRISPR系统因此被开发成真核生物基因组编辑工具(JinekMetal.,2012；WangHetal.,2013)，并广泛应用于不同的真核生物和细菌(DoudnaJAetal.,2014；SanderJDetal.,2014；HsuPDetal.,2014；SelleKetal.,2015)。
在过去的十年中，各个实验室在一些古菌模式种里建立起了有效的遗传操作体系和工具(LeighJAetal.,2011)。尽管如此，古菌的遗传学研究因其独特的生长条件，生长缓慢以及对大多数抗生素不敏感等因素而仍然显得非常具有挑战性的(ValentineDLetal.,2007)。因此，居于CRISPR系统的基因组编辑方法在古菌中的应用仍然有待研究和开发。
目前基于CRISPR系统的基因组编辑工作所利用的均是II型CRISPR系统即CRISPR/Cas9体系，而CRISPR/Cas9系统也存在诸多局限性。首先，CRISPR/Cas9可能存在脱靶效应，已经有研究发现Cas9蛋白可能会容许crRNA与靶标序列之间存在一定程度的错配，而这些错配出现的数量和位置必然会影响到基因编辑的特异性。其次，利用外源的CRISPR/Cas9系统，需要对Cas9蛋白进行适用于宿主细胞的优化改造，并需同时在细胞内表达Cas9蛋白和sgRNA(向导RNA)才能发挥作用，使用程序比较复杂。另外，在一些生长条件极端的生物中，胞内环境可能会影响到Cas9蛋白的活性，从而限制了CRISPR/Cas9系统的应用。
本发明针对以上问题，我们提出了利用原核生物自身的CRISPR系统做基因组编辑。该方法同时利用了CRISPR系统的DNA干涉活性和同源重组，因此极大程度上避免了脱靶效应，增强了编辑特异性。该方法只需要构建一个同时携带人工CRISPR簇和供体DNA的编辑质粒，流程简单，操作周期短。该方法利用的是原核生物自身的CRISPR系统，所有含有内源CRISPR-Cas系统的细菌和古菌均适用。
硫化叶菌是CRISPR研究的模式生物，关于硫磺矿硫化叶菌S.solfataricusP2和冰岛硫化叶菌S.islandicusREY15A中I型和III型CRISPR-Cas系统的研究表明其均具有DNA和(或)RNA干涉活性(ManicaAetal.,2011；ManicaAetal.,2013；ZebecZetal.,2014；GudbergsdottirS.,2011)。我们发现在冰岛硫化叶菌S.islandicusREY15A中I-A型和III-BCmr-α均具有DNA干涉活性(DengLetal.,2013)。“CCN”和“TCN”是冰岛硫化叶菌I-ACRISPR系统DNA干涉活性所需的两个PAMs(protospacer-adjacentmotifs)(LillestolR.Ketal.,2009)。而III-B型CRISPR所介导的DNA干涉活性是不需要PAM序列的，但其依赖于转录，并需确保crRNA5’端repeat序列与protospacer上游序列错配(DengLetal.,2013)。
发明内容
本发明的目的在于提供含有CRISPR-Cas系统的原核生物在内源编辑原核生物基因组中的应用，所述的原核生物为含有内源I型或III型CRISPR-Cas系统，或同时含有内源I型和III型CRISPR-Cas系统的细菌。
本发明的另一个目的在于提供一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，通过构建一个同时携带人工CRISPR簇和供体DNA的编辑质粒，在内源CRISPR系统对基因组发生DNA干涉后通过同源重组达到对基因组的编辑。
为了达到上述目的，本发明采取以下技术措施：
本发明所要保护的内容包括，含有CRISPR-Cas系统的原核生物在内源编辑原核生物基因组中的应用；内源CRISPR-Cas系统在编辑原核生物基因组中的应用。
一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，包括以下步骤：
1)构建基因组编辑质粒：
在原核生物基因组上拟编辑区域选取一段序列作为protospacer即靶标位点，根据protospacer设计两条反向互补的引物，其序列分别为正向引物：5’-AAAG-Nn-3’，反向引物：5’-TAGC-N’n-3’，其中Nn和N’n为反向互补序列，N和N’表示碱基A、T、G或C，n表示protospacer的碱基个数；将上述两条引物退火形成具有粘性末端的双链DNA，即spacer片段；人工CRISPR载体pSe-Rp经过限制性内切酶BspMI酶切处理，然后与具有粘性末端的spacer片段酶连，得到能产生成熟的crRNA的人工CRISPR质粒(pAC)；再将包含突变序列和与宿主细胞基因组上靶标位点两端同源的供体DNA片段插入到上述pAC质粒上，得到基因组编辑质粒(pGE)；
所述的供体DNA片段由左右两条同源片段与中间设计的突变序列通过SOE-PCR的方法扩增而成。
2)突变株的获得：pGE质粒电转入原核生物感受态细胞后，质粒上的人工CRISPR簇转录出pre-crRNA，pre-crRNA在细胞内被加工成成熟的crRNA；crRNA与细胞内源的CRISPR-Cas蛋白形成crRNP复合体，通过crRNA与宿主细胞基因组上的目标DNA链配对来识别靶标位点进行切割；随后质粒上的供体DNA片段与靶标位点两侧序列发生同源重组，进而得到基因组编辑突变株。
以上所述的步骤中，所述的原核生物为含有内源I型或III型CRISPR-Cas系统，或同时含有内源I型和III型CRISPR-Cas系统的细菌或古菌，包括但不限于冰岛硫化叶菌(优选原核冰岛硫化叶菌S.islandicusREY15A)，为所有含有内源CRISPR-Cas系统的细菌和古菌，例如：大肠杆菌Escherichiacoli，表皮葡萄球菌Staphylococcusepidermidis，腐败希瓦菌Shewanellaputrefaciens，嗜热细菌Thermusthermophilus，嗜热链球菌Streptococcusthermophilus，强烈火球菌Pyrococcusfuriosus，嗜酸热硫化叶菌Sulfolobusacidocaldarius，硫磺矿硫化叶菌Sulfolobussolfataricus，热自养甲烷热杆菌Methanothermobacterthermautotrophicus，盐富饶菌Haloferaxvolcanii，黑胫病菌Pectobacteriumatrosepticum，白喉棒状杆菌Corynebacteriumdiphtheriae等。
以上所述的基因组编辑包括但不限于缺失、插入和点突变。
与现有技术相比，本发明具有以下优点：
本发明提供的利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，只需构建一个同时携带人工CRISPR簇和供体DNA的编辑质粒，在内源CRISPR系统对基因组发生DNA干涉后通过同源重组达到对基因组的编辑。其最大的优点在于：应用宿主范围广，所有含有内源CRISPR-Cas系统的细菌和古菌均可操作；可用于多种编辑方式，缺失、插入和点突变等均可操作；更高的编辑效率，筛选阳性率高，背景低；流程简单，时间周期短，大大减轻原核生物基因组编辑的工作量。
附图说明
图1为本发明利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法的原理示意图。
图2中A为实施例1中利用冰岛硫化叶菌S.islandicusREY15A内源CRISPR系统对其lacS基因进行精准缺失突变设计示意图；
图2中B为实施例1中X-gal染色分析比较野生型与转化子示意图；
图2中C为实施例1中PCR检测转化子是否为43bp缺失突变株示意图；
图2中D为实施例1中DNA测序分析比较野生型和突变型lacS示意图。
图3中A为本发明实施例2中利用冰岛硫化叶菌S.islandicusREY15A内源III-B型CRISPR系统对其Cmr-2α蛋白的C端进行6×His标签插入突变设计示意图；
图3中B为实施例2中DNA测序比较野生型cmr-2α和携带6×His标签突变型cmr-2α示意图；
图3中C为实施例2中用His鼠抗westernblot鉴定携带6×His标签的Cmr-2α蛋白示意图；
图3中D为实施例2中在Cmr2α-His菌株中用携带6×His标签的Cmr-2α蛋白做共纯化示意图。
图4为实施例2中pGE-2α-His电转Δcas3/cas7和Δcas3感受态细胞，并PCR检验转化子示意图；
图4中A为实施例2中PCR所用引物的设计示意图；
图4中B为实施例2中两个缺失菌株各12个转化子PCR验证电泳图。
图5为本发明实施例3中Cmr-2α(SiRe-0894),Cmr-2β(SiRe-0598)和它们的同源蛋白PyrococcusfuriosusPF1129进行N端序列比对示意图；
图5中A为实施例3中四个保守的氨基酸选为拟突变位点示意图；
图5中B为实施例3中利用冰岛硫化叶菌S.islandicusREY15A内源I-A型CRISPR系统对其Cmr-2α蛋白HDdomain进行多位点突变设计示意图；
图5中C为实施例3中DNA测序显示Cmr-2α蛋白HDdomain三种突变型示意图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别说明，实施例中的实验操作均按常规实验条件或按照材料、试剂制造厂商说明书建议的条件。
实施例1：
一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，包括以下步骤：
本实施例利用冰岛硫化叶菌S.islandicusREY15A内源(I-A型CRISPR系统和III-B型CRISPR系统)CRISPR系统对其lacS基因进行精准缺失突变为例进行说明：
1.编辑质粒的构建
(1)在冰岛硫化叶菌S.islandicusREY15Aβ-半乳糖苷酶基因lacS(SIRE_RS11295)上选取+933至+972共40个碱基作为protospacer，其反向互补序列为5’-AGTGTAGTAATTAACACCAATCCAGTCTAACCTACCCCTT-3’，其紧邻一个CCT-PAM(ProtospacerAdjacentMotif)，因此能被I-A型CRISPR系统target；同时又由于crRNA5’端序列与对应的targetsite序列错配，所以也能同时被III-B型CRISPR系统target。基于这个protospacer设计两条引物(LacS-E-SpF/LacS-E-SpR)(表1)，两条引物通过退火生成两端含有粘性末端的spacer片段；
表1.本发明所用到引物序列
上表中黑色下划线标识的为限制性酶切位点，形成spacer粘性末端的四个碱基被黑色加粗显示；Cmr-2αHDdomain突变位点被斜体字体标识。
(2)人工CRISPR载体pSe-Rp(Peng,2015)经BspMI酶切处理，酶切产物与上述spacer片段酶连，得到人工CRISPR质粒；
(3)设计两条在lacS基因上缺失43bp的SOEPCR引物(LacS-E-SOEF/LacS-E-SOER)和分别带有SalI和NotI酶切位点的两条引物(LacS-E-SalIF/LacS-E-NotIR)(表1)，用SOEPCR的方法得到用于同源重组的供体DNA片段；
(4)供体DNA片段和上述人工CRISPR质粒分别用SalI和NotI酶切，然后酶连转化，得到编辑质粒pGE-lacS1；
2.突变株的筛选
(1)500ngpGE-lacS1质粒电转入冰岛硫化叶菌S.islandicusREY15A感受态细胞，涂布SCV(0.2％sucrose,0.2％casaminoacidsplus1％vitaminsolution)固体培养基，78℃培养7天，挑取转化子于5mlSCV液体培养基培养三天；
(2)各取90μl菌液分别添加10μlX-gal(20mg/ml)78℃孵育一个小时，观察反应体系颜色；
(3)然后分别用(F1/R1和F2/R2)(表1)进行PCR检测，发现挑取的17个转化子均含有pGE-lacS1质粒，同时其中16个转化子的lacS基因发生了43bp缺失，1个为逃逸突变株，并通过引物(LacS-Seq-F/LacS-Seq-R)(表1)扩增基因组DNA测序验证，结果显示该方法的编辑效率在90％以上。
实施例2：
一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，包括以下步骤：
本发明实施例以冰岛硫化叶菌S.islandicusREY15A内源III-B型CRISPR系统对其Cmr-2α蛋白的C端进行6×His标签插入突变为例，进行说明。
1.编辑质粒的构建
(1)在冰岛硫化叶菌S.islandicusREY15A基因组上选取cmr-2α基因(SIRE_RS04505)的最后25个碱基和cmr-3α基因的前7个碱基共32个碱基作为protospacer，其反向互补序列为5’-AGTGTAGTAATTAACACCACAATCCAGTCTAACCTACCCCTT-3’，其前面没有CCN-PAM，因而只具有III-B型CRISPR的DNA干涉活性。基于这个protospacer设计两条引物(2α-His-SpF/2α-His-SpR)(表1)，两条引物通过退火生成两端含有粘性末端的spacer片段；
(2)人工CRISPR载体pSe-Rp经BspMI酶切处理，酶切产物与上述spacer片段酶连，得到人工CRISPR质粒；
(3)设计两条包含六个组氨酸编码子的SOEPCR引物(2α-His-SOEF/2α-His-SOER)和分别带有SalI和NotI酶切位点的两条引物(2α-His-SalIF/2α-His-NotIR)(表1)，用SOEPCR的方法得到用于同源重组的供体DNA片段；
(4)供体DNA片段和上述人工CRISPR质粒分别用SalI和NotI酶切，然后酶连转化，得到编辑质粒pGE-2α-His；
2.突变株的筛选
(1)500ngpGE-2α-His质粒电转入冰岛硫化叶菌S.islandicusREY15A感受态细胞，涂布SCV(0.2％sucrose,0.2％casaminoacidsplus1％vitaminsolution)固体培养基，78℃培养7天，挑取转化子于5mlSCV液体培养基培养三天；
(2)用引物(2α-Seq-F/2α-Seq-R)(表1)扩增cmr-2α基因进行测序验证，结果显示转化子为成功插入6×His标签的突变株；
(3)用含5-FOA的SCV培养基对插入突变株进行反筛，以消除编辑质粒pGE-2α-His；
3.His标签融合蛋白的共纯化与检测
3LSCV+U(尿嘧啶)培养基培养Cmr2α-His菌株，待OD600达到0.4时，收集菌体并重悬于50mMPBS缓冲液，超声波破碎后高速离心取上清，与500μlNi琼脂糖珠充分混合一小时后过柱，然后分别用20mM咪唑PBS，60mM咪唑PBS洗柱，最后用2ml200mM咪唑PBS重复洗脱Ni柱两次，SDS-PAGE后分别银染分析和用His鼠抗做westernblot检测。
检测了7个转化子，7个均为插入突变株。
4.为了进一步排除I-A型CRISPR系统在本实施例中的影响，我们用编辑质粒pGE-2α-His电转两个缺失了I型CRIPSR活性的冰岛硫化叶菌S.islandicusREY15A感受态细胞：Δcas3/cas7和Δcas3，各挑取12个转化子进行PCR验证，得到的插入突变株比例分别为11/12和12/12(图4)，进一步说明该方法成功率达到90％以上。
实施例3：
一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，包括以下步骤：
本实施例以利用冰岛硫化叶菌S.islandicusREY15A内源I-A型CRISPR系统对其Cmr-2α蛋白HDdomain进行多位点突变为例，进行说明。
1.编辑质粒的构建
(1)在冰岛硫化叶菌S.islandicusREY15A基因组上选取cmr-2α基因N端HDdomain四个保守氨基酸H/D/K/I处选取40个碱基作为protospacer，其序列为5’-CGACCCTCCTTGGAAGGCATGGGTAATTACAAGGAATATT-3’，其前面有CCA-PAM。基于这个protospacer设计两条引物(2α-HDmut-SpF/2α-HDmut-SpR)，两条引物通过退火生成两端含有粘性末端的spacer片段；
(2)人工CRISPR载体pSe-Rp经BspMI酶切处理，酶切产物与上述spacer片段酶连，得到人工CRISPR质粒；
(3)设计两条包含四个突变氨基酸编码子的SOEPCR引物
(2α-HDmut-SOEF/2α-HDmut-SOER)和分别带有SalI和NotI酶切位点的两条引物
(2α-HDmut-SalIF/2α-HDmut-NotIR)(表1)，用SOEPCR的方法得到用于同源重组的供体DNA片段；
(4)供体DNA片段和上述人工CRISPR质粒分别用SalI和NotI酶切，然后酶连转化，得到编辑质粒pGE-2αHD；
2.突变株的筛选
(1)500ngpGE-2αHD质粒电转入冰岛硫化叶菌S.islandicusREY15A感受态细胞，涂布SCV(0.2％sucrose,0.2％casaminoacidsplus1％vitaminsolution)固体培养基，78℃培养7天，挑取转化子于5mlSCV液体培养基培养三天；
(2)用引物(2α-Seq-F/2α-Seq-R)(表1)扩增cmr-2α基因进行测序验证，挑取的7转化子均为突变株，说明编辑效率为100％，由于同源重组位置的多样性，结果显示出三种突变类型，分别为只突变H/D两个氨基酸，突变H/D/K三个氨基酸以及H/D/K/I四个氨基酸均发生突变，比例为1:2:4。
实施例4：
我们利用此方法在冰岛硫化叶菌S.islandicusHVE10/4(一个广泛应用于病毒研究的菌株)中利用其I-A型CRISPR系统成功进行了lacS基因的编辑。pGE质粒的构建及操作步骤同上实施例1。值得指出的是，S.islandicusHVE10/4由于转化效率很低，在实施传统基因敲除方法时很难成功，而利用本发明提供的内源CRISPR系统基因组编辑方法，挑取的转化子均为突变株，编辑效率达到了100％。
本发明保护范围不限于上述实施例。
尽管上文已经用一般性说明及具体实施例对本发明做了详尽的描述，但在本发明基础上作一些修改或改进，这对本领域技术人员而言是一望而知的，毋庸赘述。因此，在未偏离本发明主旨的基础上做的或多或少的修改或改进，均属于本发明要求保护的范围。
Claims (3)
1.含有CRISPR-Cas系统的原核生物在内源编辑原核生物基因组中的应用。
2.根据权利要求1所述的应用，所述的原核生物为含有内源I型或III型CRISPR-Cas系统，或同时含有内源I型和III型CRISPR-Cas系统的细菌或古菌。
3.根据权利要求1所述的应用，其方法包括：
1）构建基因组编辑质粒：
在原核生物基因组上拟编辑区域选取一段序列作为protospacer即靶标位点，将其序列克隆到两个CRISPR-Repeat序列之间，得到人工CRISPR质粒，再将包含突变序列和与宿主细胞基因组上靶标位点两端同源的供体DNA片段插入到上述人工CRISPR质粒上，得到基因组编辑质粒；
2）突变株的获得：pGE质粒电转入原核生物感受态细胞，在内源CRISPR-Cas系统对靶标位点进行DNA干涉之后，质粒上的供体DNA片段与靶标位点两侧序列发生同源重组，进而得到基因组编辑突变株。
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| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US12359218B2 (en) | 2017-07-28 | 2025-07-15 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
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| US12157760B2 (en) | 2018-05-23 | 2024-12-03 | The Broad Institute, Inc. | Base editors and uses thereof |
| US12281338B2 (en) | 2018-10-29 | 2025-04-22 | The Broad Institute, Inc. | Nucleobase editors comprising GeoCas9 and uses thereof |
| US12351837B2 (en) | 2019-01-23 | 2025-07-08 | The Broad Institute, Inc. | Supernegatively charged proteins and uses thereof |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US12281303B2 (en) | 2019-03-19 | 2025-04-22 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US12031126B2 (en) | 2020-05-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN114231552A (zh) * | 2020-09-09 | 2022-03-25 | 中国科学院微生物研究所 | 一种新的I型CRISPR/Cas系统及其应用 |
| CN114231552B (zh) * | 2020-09-09 | 2024-10-01 | 中国科学院微生物研究所 | 一种新的I型CRISPR/Cas系统及其应用 |
| CN114657177A (zh) * | 2020-12-24 | 2022-06-24 | 中国科学院微生物研究所 | 一种利用I型CRISPR-Cas系统同时实现基因编辑和转录调控的方法 |
| CN112746075A (zh) * | 2021-02-02 | 2021-05-04 | 山东大学 | 诱导型内源CRISPR-Cas系统的构建方法及其在多基因同时编辑中的应用 |
| CN116064631A (zh) * | 2022-11-17 | 2023-05-05 | 山东大学 | 一种识别并删除大片段非必需基因区的质粒的构建方法及其在基因编辑中应用 |
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