CN116083576B - 一种基于CRISPR/Cas12a的KRAS热点基因突变检测系统和方法 - Google Patents
一种基于CRISPR/Cas12a的KRAS热点基因突变检测系统和方法 Download PDFInfo
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Abstract
本发明涉及一种基于CRISPR/Cas12a的KRAS热点基因突变检测系统和方法，所述检测系统包括针对KRAS热点突变基因的重组酶聚合酶扩增RPA引物、crRNA、LbaCas12a和单链DNA荧光报告基团。本发明对仪器设备需求低、操作简便、通用性强且准确性高；利用本发明的检测系统能够快速实现对KRAS热点基因突变检测，且能够高特异性地鉴定SNV突变。
Description
技术领域
本发明属于基因检测领域，特别涉及一种基于CRISPR/Cas12a的KRAS热点基因突变检测系统和方法。
背景技术
鼠类肉瘤病毒癌基因(kirsten rat sarcoma viral oncogene,KRAS)是细胞信号通路的关键基因之一，长约35kb，位于12号染色体，是RAS基因家族成员之一。KRAS基因编码的RAS蛋白与GTP结合后具有GTP酶活性，可激活细胞内外信号通路，从而调控细胞生长表达等重要生命过程。KRAS基因发生突变时无需EGFR(epidermal growth factor receptor)接受信号，能够自动活化该通路并启动下游的信号转导，导致细胞内信号传导紊乱，细胞增殖失控。KRAS基因突变发生在肿瘤早期，且原发灶和转移灶的KRAS基因高度保持一致。有研究表明，90％的KRAS基因突变发生在2号外显子的12密码子和13密码子位点，其最常见高频突变类型为点突变，如c.35G>T，c.34G>T，c.38G>A，c.35G>A，c.35G>C，c.34G>C等。上述突变破坏了KRAS蛋白内在的GTPase活性，从而使得KRAS蛋白处于持续活性状态。一般认为，KRAS基因状态不会因治疗而发生变化。已有研究表明，KRAS基因突变分析是非小细胞肺癌(NSCLS)和结直肠癌(CRC)治疗和诊断的重要环节，KRAS基因状态会影响相关肿瘤治疗过程中的药效。因此，KRAS基因突变状态的检测对于肿瘤诊断和监测，还有靶向药物的选择都具有重要意义。
目前临床应用的KRAS基因突变检测方法主要有扩增阻滞突变系统PCR(Amplification Refractory Mutation System PCR，ARMS-PCR)、流式技术的磁珠乳液扩增方法(bead,emulsion,amplification and magnetic,BEAMing)、数字化PCR(DigitalPCR，dPCR)和高通量测序(next-generation sequencing，NGS)等，但上述方法存在操作复杂、设备昂贵且人员需专业培训等问题。因此，亟需提供一种操作简单、成本低廉，且灵敏度高、特异性强的KRAS热点基因突变检测方法，对于相关肿瘤的早期检测、用药指导和预后监测将具有重要意义。
发明内容
本发明所要解决的技术问题是提供一种基于CRISPR/Cas12a的KRAS热点基因突变检测系统和方法。该检测系统对仪器设备需求低、操作简便、通用性强且准确性高；能够快速实现对KRAS热点基因突变检测，且能够高特异性地鉴定SNV突变。
本发明提供了一种基于CRISPR/Cas12a的KRAS热点基因突变检测系统，所述检测系统包括针对KRAS热点突变基因的重组酶聚合酶扩增(Recombinase PolymeraseAmplification，RPA)引物、crRNA、LbaCas12a和单链DNA荧光报告基团；其中，所述crRNA为特异性KRAS热点基因突变G12C-crRNA，序列如SEQ ID NO:1所示；所述RPA引物包括正向/反向引物G12C-F/R。
所述G12C-crRNA与原始crRNA相比带有一个额外错配碱基。所述的错配碱基是指，在待测突变位点处有与目标序列不互补的错配碱基。所述crRNA可通过构建体外转录载体并进行体外转录和纯化制得，或者直接合成。
所述G12C-F/R的序列如SEQ ID NO:2～3所示。所述G12C-F/R引物是通过相关引物设计原则筛选出来的高效特异序列，其中G12C-F序列中带有非典型PAM序列(GTTG)。
所述LbaCas12a可经重组表达纯化获得或使用NEB等公司提供的商品化LbaCas12a产品。
所述单链DNA荧光报告基团的序列如下所示：5'-6-FAM-TTTTT-IABkFQ-3'。
本发明还提供了一种基于CRISPR/Cas12a的KRAS热点基因突变检测方法，包括：
采用所述RPA引物对待测核酸样本进行RPA扩增，得到RPA扩增产物；将crRNA、RPA扩增产物、LbaCas12a、单链DNA荧光报告基团混合于反应体系中进行反应；反应产物通过荧光检测获得检测结果。
所述反应体系为20μL，0.5μl的crRNA、1.0μl的LbaCas12a、2μl 10×NEBufferTM2.1、2μl单链DNA荧光报告基团、5μl的RPA扩增产物和9.5μl的水。
所述反应条件为37℃10～60分钟。
所述待测核酸样本可以是从临床样本中抽提的核酸，或以其他核酸检测手段中样本处理方法处理过的样品。
所述荧光检测所用装置可为任何能在FAM荧光通道上进行荧光激发并检测的荧光检测装置，或通过蓝光照射进行裸眼可视化观察。
有益效果
(1)本发明与现有其它检测技术相比，所述方法只需恒温装置和简易荧光读取装置或裸眼直接观察，操作简单，检测速度快，在KRAS热点基因突变检测中表现出良好性能，能够对低丰度的KRAS热点基因突变位点进行准确检测，具有良好应用前景。
(2)本发明方法中通过RPA的高效扩增能力产生大量扩增产物，上述扩增产物可被Cas12a所特异识别，识别过程中需相应PAM位点，利用非典型PAM位点方式可解决部分靶标突变序列处无PAM位点的问题，大大增加了本发明方法的通用性；同时CRISPR/Cas12a的高特异性和自我放大能力又进一步提高了传感性能，特别是通过crRNA错配碱基方式可实现高效区分SNV位点的目的，从而使所述检测方法具有准确性好、灵敏度高、选择性强、抗干扰能力强、重复性好等优点。
附图说明
图1为应用非典型PAM位点进行CRISPR/Cas12a检测的可行性分析结果。
图2为KRAS热点基因突变G12C检测的荧光信号强度和裸眼观察结果。
图3为KRAS热点基因突变G12C检测灵敏度(A)和选择性(B)分析结果图。
图4为KRAS热点基因突变G12C检测特异性分析结果图。
图5为CRISPR/Cas12a荧光检测方法的临床应用。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，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。
首先简单阐述本发明的机理：本发明中使用的是V型CRISPR系统，其中效应蛋白主要为Cas12家族，该类核酸酶普遍具有非特异性的ssDNA切割活性。当Cas12效应蛋白在特征性crRNA引导下靶向目标DNA序列后，会激活其顺式反应活性和反式切割活性。通过加入ssDNA荧光报告探针，当样本中存在靶标DNA序列时，Cas12反式切割活性被激活，探针被切断释放荧光，从而实现目标基因序列的检测。
进一步实验发现，当crRNA与靶标序列间存在碱基错配时会对反式切割活性造成影响，在特定位置的错配会导致切割活性明显减弱乃至丧失。基于此，可实现对SNV位点的检测。
下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂商所建议的实验条件。所使用的材料和试剂等，如无特殊说明，均为可通过商业途径购得的常规产品。过商业途径购得的常规产品。
实施例1
CRISPR/Cas12a荧光检测平台的建立
本实施例提供了一种基于CRISPR/Cas12a的KRAS热点基因突变检测方法，以12密码子中最为常见G12C点突变为例进行实验。
1.序列设计
12密码子中存在G12C突变与其对应野生型两种类型。
12密码子中G12C突变参考序列如下(如SEQ ID NO:4所示)：
5’-ATGACTGAATATAAACTTGTGGTAGTTGGAGCTTGTGGCGTAGGCAAGAGTGCCT TGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGATT CCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGA-3’
12密码子中G12C突变对应野生型参考序列如下(如SEQ ID NO:5所示)：
5’-ATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCT TGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGATT CCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGA-3’
其中划线部分碱基为KRAS基因G12C突变位点。
针对KRAS基因2号外显子12密码子上的G12C突变设计特异性G12C-crRNA序列，与原始crRNA不同，该序列还带有一个额外错配碱基以有效实现SNV位点的特异检测和区分，参考序列如下(如SEQ ID NO:1所示)：
5’-UAAUUUCUACUAAGUGUAGAUGAGCUUGUGGCGAAGGCAAG-3’
其中划线部分碱基为G12C-crRNA引入的一个额外错配碱基。
构建G12C-crRNA的转录载体，通过T7高产量转录试剂盒(Thermo FisherScientific)转录制备。制备后的crRNA经RNAClean&ConcentratorTM-5(Zymo Research)纯化。
针对KRAS基因2号外显子12密码子上的G12C突变及其对应野生型序列，设计合成扩增上述目的片段的RPA正向和反向引物，因T790M突变位点附近无PAM序列，无法进行Cas酶识别切割，故使用原序列中存在的非典型PAM序列(GTTG)替代典型PAM序列(TTTN)。
具体的，G12C突变位点的RPA引物正向序列为(如SEQ ID NO:2所示)：GACTGAATATAAACTTGTGGTAGTTGGAGC；反向引物序列为(如SEQ ID NO:3所示)：GTCGAGAATATCCAAGAGACAGGTTTCTCC。其中划线部分为RPA正向引物中带有的非典型PAM序列。
2.RPA扩增
待测核酸样本处理方法为取样本溶液10μL进行RPA扩增，所得的扩增产物即处理后的核酸样本，本实施例中涉及待测核酸样品为人工合成的分别含有KRAS基因2号外显子12密码子上的G12C突变及其对应野生型序列的质粒DNA。
RPA反应体系：50μl体系中，29.5μL rehydration buffer，0.6μL正向引物和反向引物(10μM)，10μL待测核酸样本模板、6.8μL水，将上述体系混合后加入2.5μL醋酸镁(280mM)，37℃反应20min。
3.CRISPR荧光检测反应体系
单链DNA荧光报告基团的设计合成：5’端为FAM荧光基团的5个T碱基组成的单链双淬灭DNA荧光探针5'-6-FAM-TTTTT-IABkFQ-3'。
将上述G12C-crRNA体外转录产物、处理后的核酸样本、LbaCas12a、单链DNA荧光报告基团以适当比例混合于适当体系中进行反应。
反应体系为：20μl体系中，0.5μl的crRNA(10μM)、1.0μl的LbaCas12a(10μM)、2μl10×NEBufferTM 2.1、2μl单链DNA荧光报告基团(10μM)、5μl处理后的核酸样本和9.5μl的水。反应体系在37℃反应10～60min。
反应产物可通过荧光检测装置检测，使用波长485nm的激发光激发荧光，在波长535nm处检测荧光强度以获得检测结果；或通过蓝光照射直接通过裸眼进行可视化观察判定结果。
验证使用非典型PAM序列(GTTG)替代典型PAM序列(TTTN)的可行性，50μl体系中RPA在37℃条件下反应20min，在20μl体系中进行Cas12蛋白切割反应，酶标仪上37℃反应30min，每隔1min读取反应荧光信号。结果如图1所示，表明非典型PAM序列也可有效介导LbaCas12a识别并切割，且在30min时的切割效果可等同于典型PAM序列。
检测结果显示，反应时间30min时，本发明所述的检测系统可有效区分KRAS基因2号外显子12密码子上的G12C突变及其对应野生型序列，如图2所示。
实施例2
KRAS热点基因突变CRISPR/Cas12a荧光检测体系的灵敏度
用CRISPR/Cas12a检测10倍倍比稀释的KRAS热点基因点突变G12C核酸样本，浓度范围为1aM到10,000aM。灵敏度和选择性分析结果显示，反应时间30min时，本发明所述的检测系统对样本的检测限可低至100aM，可高选择性地从野生型样本中检出突变频率为0.02％低丰度突变型样本，如图3所示。
实施例3
KRAS热点基因突变CRISPR/Cas12a荧光检测体系的特异性
以KRAS常见热点基因突变(G12A、G12S、G12R、G12C、G12D、G12V和G13D)核酸样本为模板，评价KRAS热点基因突变CRISPR/Cas12a荧光方法的特异性，其中有显著荧光值变化的为阳性，无明显荧光值变化的为阴性。
特异性分析结果显示，仅G12C进行CRISPR/Cas12a荧光检测时有显著荧光值变化，其他KRAS热点基因突变均无荧光值，说明本发明所述的检测系统具有良好的特异性，如图4所示。
实施例4
KRAS热点基因突变CRISPR/Cas12a荧光检测体系用于临床样本检测
为评价CRISPR/Cas12a荧光检测方法检测临床标本的性能，我们将收集的4份KRAS-G12C突变样本和3份KRAS-G12C野生型样本使用CRISPR/Cas12a荧光检测体系进行检测，同时以qPCR方法作为对照试验。
结果如图5所示，CRISPR/Cas12a可有效区分KRAS-G12C突变样本和其对应野生型样本，且其检测结果和qPCR方法结果完全一致，说明本发明所述的检测系统可有效用于临床样本检测，且检测时间极大缩短，50min即可完成检测。
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。
Claims (1)
1.一种基于CRISPR/Cas12a的KRAS热点基因突变检测系统，其特征在于：所述检测系统包括针对KRAS热点突变基因的重组酶聚合酶扩增RPA引物、crRNA、LbaCas12a和单链DNA探针；其中，所述crRNA为特异性KRAS热点基因突变G12C-crRNA，序列如SEQ ID NO:1所示；所述RPA引物包括正向/反向引物G12C-F/R，序列如SEQ ID NO:2～3所示；所述单链DNA探针的序列如下所示：5'-6-FAM-TTTTT-IABkFQ-3'。
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