CN117701682A - 基于发夹探针激活CRISPR-Cas12a系统的方法及其应用 - Google Patents
基于发夹探针激活CRISPR-Cas12a系统的方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于发夹探针激活CRISPR‑Cas12a系统的方法，包括以下步骤：制备发夹探针；将crRNA和Cas12a蛋白混合后，加入反应缓冲液、发夹探针、F‑Q探针、核糖核酸酶抑制剂混匀，最后加入靶miRNA，孵育后终止酶反应；所述发夹探针的环部区域为miRNA的识别域，茎部区域为crRNA的结合域，所述发夹探针中无PAM序列；当靶miRNA与所述发夹探针的环部区域杂交后，引发crRNA与发夹探针的茎部区域相结合，从而激活CRISPR‑Cas12a系统的顺式和反式剪切活性。本发明通过设计一种不含PAM序列的发夹探针作为CRISPR‑Cas12a的触发底物，在靶miRNA存在时，通过CRISPR/Cas12a系统激活的反式剪切活性剪切荧光信号探针，可实现一步反应对miRNA的快速、灵敏检测。
Description
技术领域
本发明涉及一种基于发夹探针激活CRISPR-Cas12a系统的方法及其应用，属于基因检测及分子诊断领域。
背景技术
MicroRNA(miRNA)是一种由18-24个核苷酸组成的短链非编码RNA，在多种癌症的发生发展中发挥着重要的作用。大量研究发现在癌症中miRNA普遍异常表达或特定类型的miRNA异常表达，以及参与癌症对各种治疗耐药性的形成；且证明了miRNA在癌症诊断、药物疗效监测及预后评估中作为生物标志物的潜力，因此，快速、灵敏、准确检测miRNA具有重要的临床价值。目前，miRNA定量检测方法主要采用实时荧光定量PCR(qRT-PCR)技术，但由于miRNA序列短，通常采用茎环法或者加尾法对其进行逆转录获得cDNA之后，再结合荧光定量PCR对其进行检测，所以该方法存在引物设计困难、操作步骤繁琐和耗时长等缺点。近年来，多种等温核酸扩增技术被引入miRNA检测方法的开发，虽在一定程度上满足了短链miRNA检测的需求，但这些方法在准确性上难以与传统RT-PCR技术相媲美，因此，未能发展为临床适用的miRNA检测方法。
CRISPR系统已成为基因编辑、转录调控和分子诊断的强大工具。其中，CRISPR-Cas12a系统是最具影响力和最有前途的CRISPR-Cas系统之一。CRISPR-Cas12a系统可对剪切靶点上游含特定TTTN序列(PAM序列)的双链DNA(dsDNA)进行识别和顺式剪切活性，同时启动高效的反式剪切活性，即无差别剪切单链DNA(ssDNA)。dsDNA激活CRISPR-Cas12a系统的顺式/反式剪切活性机制如下：首先，Cas12a蛋白的氨基酸残基通过氢键和范德华力与dsDNA的PAM序列相结合。随后，Cas12a蛋白的解旋酶活性被激活，导致dsDNA不稳定并解旋，然后crRNA的可编程间隔序列与不稳定的dsDNA上靶标链(TS)杂交，进而激活CRISPR-Cas12a系统的顺式/反式剪切活性。由于传统的dsDNA激活模式需要PAM序列，因此，极大限制了CRISPR-Cas12a系统应用的通用性。
综上，亟待发展一种无PAM限制激活CRISPR-Cas12a系统的模式，并应用于构建快速、灵敏、准确检测miRNA的方法。
发明内容
本发明提供了一种基于发夹探针激活CRISPR-Cas12a系统的方法，利用靶miRNA与发夹探针一步杂交链置换反应即可高效激活CRISPR-Cas12a系统，由此实现免PAM序列识别的直接、快速、灵敏检测miRNA。
为达到上述目的，本发明所采用的技术方案是：一种基于发夹探针激活CRISPR-Cas12a系统的方法，包括以下步骤：制备发夹探针；将crRNA和Cas12a蛋白混合后，加入反应缓冲液、发夹探针、F-Q探针、核糖核酸酶抑制剂混匀，最后加入靶miRNA，孵育后终止酶反应；所述发夹探针的环部区域为miRNA的识别域，茎部区域为crRNA的结合域，所述发夹探针中无PAM序列；当靶miRNA与所述发夹探针的环部区域杂交后，引发crRNA与发夹探针的茎部区域相结合，从而激活CRISPR-Cas12a系统的顺式和反式剪切活性。
优选地，所述发夹探针通过95℃，5min退火制备得到。孵育后终止酶反应具体为：在37℃下孵育45min，然后在65℃下孵育10min终止酶反应。
本发明还提供了一种miR-21检测试剂盒，包含crRNA、Cas12a蛋白、反应缓冲液、发夹探针以及F-Q探针，所述发夹探针的环部含有与靶标miRNA特异性识别的核苷酸序列，所述发夹探针的茎部含有与crRNA特异性结合的核苷酸序列；所述发夹探针中不含有PAM序列。
优选地，所述发夹探针和crRNA浓度均为25nM，所述F-Q探针浓度为500nM，所述反应缓冲液为NEBuffer 3。
优选地，所述发夹探针的核苷酸序列如SEQ ID NO.2至SEQ ID NO.10任意一项所示。
优选地，所述crRNA序列如SEQ ID NO.1所示。
优选地，所述F-Q探针为修饰有FAM荧光基团和BHQ1猝灭基团的ssDNA，序列为FAM-TTATT-BHQ1，被激活的CRISPR-Cas12a系统反式剪切后，荧光信号恢复。
本发明通过设计发夹探针作为CRISPR-Cas12a系统的触发底物，实现了无PAM序列激活CRISPR-Cas12a系统的顺式剪切和反式剪切活性，同时无需繁琐逆转录等前处理步骤，实现一步反应对miRNA的快速、灵敏检测。本发明利用免PAM序列识别的发夹探针激活CRISPR-Cas12a系统，并扩展到miRNA检测的应用，具有设计简单、一步操作即可完成miRNA检测的优点。
附图说明
图1为本发明实施例中一种基于发夹探针激活CRISPR-Cas12a系统的方法的原理示意图；
图2为本发明实施例1中靶miRNA辅助发夹探针激活CRISPR-Cas12系统的PAGE结果；
图3为本发明实施例1中靶miRNA辅助发夹探针激活CRISPR-Cas12系统的荧光结果；
图4为本发明实施例1中不同发夹探针序列、不同反应缓冲液以及不同反应时间的优化结果；
图5为本发明实施例1中构建的检测方法对于miR-21检测的灵敏度；
图6为本发明实施例1中构建的检测方法对于miR-21检测的特异性。
具体实施方式
为了更好的理解本发明的实质，下面结合具体实施例和附图对本发明作进一步的阐述。
本发明的方法设计了一种无PAM序列的发夹探针作为CRISPR-Cas12a系统的触发底物，发夹探针的环部区域为识别miRNA的区域，茎部区域为crRNA的结合域。在靶miRNA存在时，靶miRNA与发夹探针的环部区域杂交，然后引发crRNA与发夹探针的茎部区域相结合，从而激活CRISPR-Cas12a系统。激活的CRISPR-Cas12a系统启动反式剪切活性剪切F-Q探针，从而对靶miRNA产生荧光信号响应，由此实现一步反应对miRNA的快速、灵敏检测。
如图1所示，本发明通过设计无PAM序列的发夹探针作为CRISPR-Cas12a系统的触发底物，当靶miRNA与发夹探针的茎部区域杂交后，引发crRNA与发夹探针的茎部区域相结合，从而激活CRISPR-Cas12a系统的顺式剪切和反式剪切活性，实现无PAM序列限制的CRISPR-Cas12a系统激活新方法。最后，利用激活的CRISPR-Cas12a系统反式剪切活性，剪切修饰有FAM荧光基团和BHQ1猝灭基团的F-Q探针，以荧光作为信号输出，实现对靶miRNA的荧光信号响应。
本发明提供了1.一种基于发夹探针激活CRISPR-Cas12a系统的方法，具体包括如下步骤：
通过退火制备发夹探针；
将crRNA和Cas12a蛋白混合后，加入反应缓冲液、制备好的发夹探针、F-Q探针以及核糖核酸酶抑制剂混匀，最后加入靶miRNA，在37℃下孵育45min，然后在65℃下孵育10min终止酶反应即得。
所述发夹探针的退火具体步骤为：将发夹探针在热循环仪中95℃下保持5-10min，在5h内冷却至4℃，取出4℃保存待用。
实施例1
一、材料
EnGen Lba Cas12a(Cpf1)、10×NEBuffer r2.1(500mM NaCl，100mM Tris-HCl，100mM MgCl2，1mg/ml重组白蛋白，pH 7.9)、10×NEBuffer 4(500m M CH3COOK，200mMTris-acetate，100mM Mg(CH3COO)2，10mM二硫苏糖醇，pH 7.9)、10×rCutsmart(500mMCH3COOK，200mM Tris-acetate，100mM Mg(CH3COO)2，1mg/ml重组白蛋白，pH 7.9)和10×NEBuffer 3(1M NaCl，500mM Tris-HCl,100mM MgCl2，10mM二硫苏糖醇，pH 7.9)购自NewEngland Biolabs(Ipswich，MA，USA)。经过HPLC纯化的crRNA、重组RNase抑制剂经过HPLC纯化的所有过核苷酸，无酶水均购自于Sangon Biotechnology Co.,Ltd(中国上海)。6×loading buffer和20-200bp marker购自Takala Biotech.Inc.(大连，中国)。PAGE凝胶快速制备试剂购自于Epizyme Biotech(中国，上海)。所有化学试剂都是分析级的，无需进一步纯化直接使用。所有的RNA反应溶液都是用无RNase的水制备的。所有的水溶液均使用超纯水(≥18兆欧，Milli-Q,Millipore)制备。
二、仪器
所有荧光光谱均使用荧光分光光度计F-4700(HATACHI，日本)记录，激发波长为490nm，发射波长为520nm；入口和出口的狭缝均为5nm处。DNA悬液用NanoDrop 1000分光光度计(Thermo Scientific,USA)定量。凝胶电泳分析在Bio-Rad电泳分析仪上进行。凝胶图像来自Bio-Rad ChemDoc XRS成像系统(Bio-Rad，USA)。
三、检测方法的构建
将表1中合成的寡核苷酸序列中加入无酶水，漩涡震荡3min，4℃静止3min，重复三次。将发夹探针H8-H16(100μM)浓度稀释至250nM；crRNA(100μM)稀释至250nM；将F-Q探针(100μM)浓度稀释至5μM。发夹探针H8-H16(250nM)通过退火形成发夹结构，其中发夹探针H8-H16的核苷酸序列如SEQ ID NO.2至SEQ ID NO.10所示；将crRNA和Cas12a蛋白混合后，加入反应缓冲液、发夹探针、F-Q探针、核糖核酸酶抑制剂混匀，最后加入靶miRNA，在37℃下孵育45min，然后在65℃下孵育10min终止酶反应，完成检测体系(Cas12a蛋白、crRNA和发夹探针浓度均为25nM，F-Q探针浓度为500nM)的构建。
表1实施例中涉及的DNA及RNA序列
四、天然聚丙烯酰胺凝胶(PAGE)电泳
制备12％天然聚丙烯酰胺凝胶，验证在miR-21为靶标存在时，是否能够激活CRISPR-Cas12a系统的反式剪切活性。
电泳于1×TBE缓冲液(2mM EDTA,89mM三硼酸，pH 8.3)中进行，在100V恒压下电泳45min，再将凝胶浸入新配制的染色液(1×TBE缓冲液60mL，含3μL GoldView I)中30min，然后使用Bio-Rad ChemDoc XRS成像系统对凝胶进行成像，其结果如图2所示。
表2 PAGE电泳的体系构成
| miR-21 PAGE体系 | 总体积20μL | 终浓度 |
| H12 | 2μL | 500nM |
| crRNA | 2μL | 500nM |
| Cas12a | 2μL | 500nM |
| Reporter-2 | 2μL | 1μM |
| miR-21 | 2μL | 500nM |
| RNase inhibitor | 1μL | 5KU |
| 10×NEBuffer 3 | 2μL | |
| RNase-free water | 7μL |
五、本发明检测方法的发夹探针、反应缓冲液和反应时间的优化。
(1)溶链：将所有上述公司合成的寡核苷酸链都按照说明书指示加入指定量无酶水，涡旋震荡3min，在4℃下静置3min，重复3次。
(2)采用前述构建的检测方法分别对试验组和对照组进行检测，检测结果如图3和图4所示。检测结果说明单独发夹探针存在时，荧光背景信号极低，当靶miRNA存在时，荧光响应信号显著增大。
在优化发夹探针和优化反应缓冲液时，实验组的相应条件进行改变，实验组和对照组分别设计三个平行组。发夹探针优化的组别分别为H8-H16九种不同序列发夹探针，反应缓冲液优化的组别分别为NEBuffer r2.1、NEBuffer 4、rCutsmart、和NEBuffer3，其中对照组不添加靶miRNA，实验组添加靶miRNA。反应时间优化则分别在0min、15min、30min、45min、60min和75min六个时间点测靶miRNA存在和不存在时的荧光响应信号。根据实验结果的信噪比可得最佳发夹探针为H12，最佳反应缓冲液为NEBuffer 3，最佳反应时间为45min。
(3)荧光检测：先用酒精和ddH2O清洗荧光比色皿，仪器参数按照二、中设置。之后用荧光分光光度计检测荧光。
表3荧光检测的体系构成
六、本发明检测方法的灵敏度和特异性考察
(1)溶链：将上述所有公司合成的寡核苷酸链都按照说明书指示加入指定量无酶水，涡旋震荡3min，在4℃下静置3min，重复3次。
(2)靶miRNA浓度的梯度稀释：从溶链得到的100μM浓度稀释至20μM，之后逐管稀释得到分别为15nM、10nM、5nM、1nM、500pM、100pM、50pM的浓度。
(3)采用前述构建的检测方法进行灵敏度实验。灵敏度实验加入不同浓度的靶miRNA引发反应，检测结果如图5所示。
表4灵敏度实验的体系构成
| miR-21检测体系 | 总体积100μL | 终浓度 |
| H12 | 10μL | 25nM |
| crRNA | 10μL | 25nM |
| Cas12a | 10μL | 25nM |
| F-Q探针 | 10μL | 500nM |
| miR-21 | 10μL | 梯度稀释的不同浓度 |
| RNase inhibitor | 1μL | 5KU |
| 10×NEBuffer 3 | 10μL | |
| RNase-free water | 49μL |
(4)采用前述构建的检测方法进行特异性实验。特异性实验则加入靶miR-21和其它五种不同的干扰miRNA实验组和对照组分别设计三个平行组，检测结果如图6所示。
表5特异性实验的体系构成
从图5和图6可以看出，本发明可检测出靶miR-21的最低浓度为50pM，展现出优异的灵敏度，同时还具有较高的特异性。
本发明的发夹探针，摆脱了CRISPR-Cas12a系统对含PAM序列的dsDNA识别的限制，扩展了其应用的通用性和范围，仅需要一步miRNA与发夹探针杂交链置换反应即可激活CRISPR-Cas12a系统，在灵敏度和特异性方面展现出巨大的优势。
以上仅为本发明的实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均包含在申请待批的本发明的权利要求范围之内。
Claims (10)
1.基于发夹探针激活CRISPR-Cas12a系统的方法，其特征在于，包括以下步骤：
制备发夹探针；
将crRNA和Cas12a蛋白混合后，加入反应缓冲液、发夹探针、F-Q探针、核糖核酸酶抑制剂混匀，最后加入靶miRNA，孵育后终止酶反应；
所述发夹探针的环部区域为miRNA的识别域，茎部区域为crRNA的结合域，所述发夹探针中无PAM序列；当靶miRNA与所述发夹探针的环部区域杂交后，引发crRNA与发夹探针的茎部区域相结合，从而激活CRISPR-Cas12a系统的顺式和反式剪切活性。
2.根据权利要求1所述方法，其特征在于：所述发夹探针通过95℃，5-10min退火制备得到。
3.如权利要求1所述方法，其特征在于，孵育后终止酶反应具体为：在37℃下孵育45min，然后在65℃下孵育10min终止酶反应。
4.一种miR-21检测试剂盒，其特征在于：包含crRNA、Cas12a蛋白、反应缓冲液、发夹探针以及F-Q探针，所述发夹探针的环部含有与靶标miRNA特异性识别的核苷酸序列，所述发夹探针的茎部含有与crRNA特异性结合的核苷酸序列；所述发夹探针中不含有PAM序列。
5.根据权利要求4所述miR-21检测试剂盒，其特征在于：所述发夹探针和crRNA浓度均为25nM，所述F-Q探针浓度为500nM。
6.根据权利要求4所述miR-21检测试剂盒，其特征在于：所述反应缓冲液为NEBuffer3。
7.根据权利要求4所述miR-21检测试剂盒，其特征在于：所述发夹探针的核苷酸序列如SEQ ID NO.2至SEQ ID NO.10任意一项所示。
8.根据权利要求4所述miR-21检测试剂盒，其特征在于：所述crRNA序列如SEQ ID NO.1所示。
9.根据权利要求4所述miR-21检测试剂盒，其特征在于：所述F-Q探针为修饰有荧光基团和淬灭基团的单链DNA。
10.如权利要求1-3任意一项所述基于发夹探针激活CRISPR-Cas12a系统的方法，在生化电路、DNA反应网络中对生物分子检测的应用，所述应用为非疾病与诊断上的应用。
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