CN111257297A - 一种基于Cas14a酶的RNA检测方法 - Google Patents
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Abstract
本发明是一种RNA激活Cas14a附带切割效应的方法，具体涉及分析化学、疾病快速诊断领域。利用RNA激活Cas14a的附带切割效应，切割ssDNA荧光探针，实现对RNA检测。本发明解决了Cas系统检测RNA时切割探针为RNA的问题，丰富了Cas14a的应用与检测领领域的范围。
Description
技术领域
本发明涉及一种基于Cas14a酶的RNA检测方法，其特征在于：可用于生物分析领域。
背景技术
CRISPR（Clustered regularly interspaced short palindromic repeats）被称为规律成簇间隔短回文重复，是细菌用以保护自身对抗病毒的一个系统[1]。目前发现的CRISPR相关联CRISPR-Cas系统效应蛋白分为两大类[2]。其中Cas9与tracrRNA、引导crRNA组成效应复合物切割靶DNA[3]；Cas12a不需要tracr RNA，其单独使用crRNA作为指导，向目标双链DNA中引入交错切割，一旦切割靶标，Cas12a的附带切割活性即可被激活，可以切割TTATT序列的DNA单链[4]；Cas13a是RNA引导的RNA酶，一旦RNA靶标与sgRNA相结合，Cas13a的“附带切割”活性即可被激活成为非特异性的RNase[5]。基于这种激活附带切割的性能，CRISPR-Cas系统被广泛应用于DNA、RNA和病原微生物的检测。CRISPR-Cas14酶是近期发现的一种新的CRISPR-Cas家族的蛋白,其分子量较小，具有靶向切割DNA的活性，本发明能够相应的提供一种RNA激活Cas14a“附带切割”活性实现对RNA的检测，对生物分析领域具有重要意义；
[1]Jackson, S. A.; McKenzie, R. E.; Fagerlund, R. D.; Kieper, S. N.;Fineran, P. C.; Brouns, S. J., CRISPR-Cas: Adapting to change. Science 2017,356 (6333).
[2]Shmakov, S.; Smargon, A.; Scott, D.; Cox, D.; Pyzocha, N.; Yan, W.;Abudayyeh, O. O.; Gootenberg, J. S.; Makarova, K. S.; Wolf, Y. I.; Severinov,K.; Zhang, F.; Koonin, E. V., Diversity and evolution of class 2 CRISPR-Cassystems. Nat Rev Microbiol 2017, 15 (3), 169-182.
[3]Chen, J. S.; Doudna, J. A., The chemistry of Cas9 and its CRISPRcolleagues. Nature Reviews Chemistry 2017, 1 (10).
[4]Max A. English; Luis R. Soenksen; Raphael V. Gayet; Helena de Puig;Nicolaas M. Angenent-Mari; Angelo S. Mao2; Peter Q. Nguyen; Collins, J. J.,Programmable CRISPR-responsive smart materials. Science 2019.
[5]Abudayyeh, O. O.; Gootenberg, J. S.; Essletzbichler, P.; Han, S.;Joung, J.; Belanto, J. J.; Verdine, V.; Cox, D. B. T.; Kellner, M. J.; Regev,A.; Lander, E. S.; Voytas, D. F.; Ting, A. Y.; Zhang, F., RNA targeting withCRISPR–Cas13. Nature 2017, 550 (7675), 280-284。
发明内容
本发明的目的是针对CRISPR-Cas系统检测RNA存在的问题，提供一种基于Cas14a酶的RNA检测方法，为实现上述目的，本发明采用的技术方案为：
向含有单链引导RNA(sgRNA)-Cas14a复合体和单链DNA荧光猝灭探针(F-ssDNA-Q)的溶液中加入待检测RNA,以荧光信号变化强度实现对RNA的定量与定性分析；所述sgRNA-Cas14a复合体的制备方法为混合sgRNA和Cas14a酶，在0-60oC混合孵育0-300分钟；所述sgRNA浓度为0-100mM；所述Cas14a的浓度为0-100 mM；所述sgRNA的序列为:UUC ACU GAUAAA GUG GAG AAC CGC UUC ACC AAA AGC UGU CCC UUA GGG GAU UAG AAC UUG AGU GAAGGU GGG CUG CUU GCA UCA GCC UAA UGU CGA GAA GUG CUU UCU UCG GAA AGU AAC CCUCGA AAC AAA UUC AUU UGA AAG AAU AAG GAA UGC AAC+间隔序列；所述单链DNA荧光猝灭探针(F-ssDNA-Q)的序列为F-(T)n-Q(n≥5),如n=5，序列长度为F-TTTTT-Q；所述间隔序列为含有与待检测RNA互补5-40 bp长度的序列。
附图说明
图1 不同种类microRNA的荧光强度柱状图；
图2 不同浓度miRNA-221的荧光强度柱状图；
图3 标准曲线。
具体实施方式
以下通过具体的实施例对本发明作进一步说明，有助于本领域的普通技术人员更全面的理解本发明，但不以任何方式限制本发明；
实施例1：
单链引导RNA(sgRNA)-Cas14a复合体的制备：将500 nM sgRNA、500 nM Cas14a和100nM FQ探针在25 mM NaCl, 20 mM Tris-HCl, 1 mM DTT 和10 mM MgCl2混合均匀后与30oC孵育30分钟；
sgRNA序列（红色下划线区为间隔序列）：UUC ACU GAU AAA GUG GAG AAC CGC UUC ACCAAA AGC UGU CCC UUA GGG GAU UAG AAC UUG AGU GAA GGU GGG CUG CUU GCA UCA GCCUAA UGU CGA GAA GUG CUU UCU UCG GAA AGU AAC CCU CGA AAC AAA UUC AUU UGA AAGAAU AAG GAA UGC AAC CAACAUCAGUCUGAUAACU
FQ探针序列：Fam-TTTTTTTTTTTT-BHQ1
实施例2：
RNA定性分析：将100nM的microRNA-221和microRNA-145加入实施例1中孵育好的sgRNA-Cas14和FQ溶液中，在96孔板中进行荧光检测，以荧光信号变化实现对相应RNA的定性检测，以水代替RNA作为空白对照组,,每组实验重复三次（参见图1）；
microRNA-221序列：UAGCUUAUCAGACUGAUGUUGA
microRNA-145序列：GUCCAGUUUUCCCAGGAAUCCCU
由图1可知，含有microRNA-221的实验组荧光信号剧烈增强，而micorRNA-145和空白对照组荧光信号没有变化，这表明该方法可以用于RNA的定性分析；
实施例3：
RNA定量分析：将不同浓度的microRNA-221入实施例1中孵育好的sgRNA-Cas14和FQ溶液中，在96孔板中进行荧光检测，通过不同浓度microRNA对应的荧光强度实现对相应RNA定性分析（参见图2和图3）；
由图2可知，随着microRNA-221浓度的增加，荧光信号逐步增强，由图3可知，micorRNA-221浓度的对数值和荧光信号值呈现出很好的线性关系，R2=0.99721，这表明该方法可用于RNA的定量分析。
Claims (6)
1.一种基于Cas14a酶的RNA检测方法，其特征在于：向含有单链引导RNA(sgRNA)-Cas14a复合体和单链DNA荧光猝灭探针(F-ssDNA-Q)的溶液中加入待检测RNA,以荧光信号变化强度实现对RNA的定量与定性分析。
2.按权利要求1所述的一种基于Cas14a酶的RNA检测方法，其特征在于：所述sgRNA-Cas14a复合体的制备方法为混合sgRNA和Cas14a酶，在0-60oC混合孵育0-300分钟。
3.按权利要求1所述的一种基于Cas14a酶的RNA检测方法，其特征在于：所述sgRNA浓度为0-100mM；所述Cas14a的浓度为0-100 mM。
4.按照权利1所述一种RNA激活CRISPR-Cas14a酶附带切割效应的方法，其特征在于：所述sgRNA的序列为TTCACTGATAAAGTGGAGAACCGCTTCACCAAAAGCTGTCCCTTAGGGGATTAGAACTTGAGTGAAGGTGGGCTGCTTGCATCAGCCTAATGTCGAGAAGTGCTTTCTTCGGAAAGTAACCCTCGAAACAAATTCATTTgaaaGAATGAAGGAATGCAAC+间隔序列。
5.按照权利1所述一种RNA激活CRISPR-Cas14a酶附带切割效应的方法，其特征在于：所述单链DNA荧光猝灭探针(F-ssDNA-Q)的序列为F-（T）n-Q(n≥5),如n=5，序列长度为F-TTTTT-Q。
6.按照权利5所述sgRNA序列，其特征在于：间隔序列为含有与待检测RNA互补5-40 bp长度的序列。
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| CN112831544A (zh) * | 2020-12-31 | 2021-05-25 | 华南农业大学 | 一种基于CRISPR/Cas12a系统的生物检测方法和生物检测装置 |
| CN114196752A (zh) * | 2021-12-08 | 2022-03-18 | 福州市讯刊生物科技有限公司 | 一种基于Cas14和链置换扩增的miR-21检测试剂盒及其应用 |
| CN114646759A (zh) * | 2020-12-17 | 2022-06-21 | 海南大学 | 一种新冠IgG/IgM抗体检测试纸条的制备方法 |
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