CN111363763A - 一种RNA激活Cas14a酶附带切割效应的方法 - Google Patents

一种RNA激活Cas14a酶附带切割效应的方法 Download PDF

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CN111363763A
CN111363763A CN202010242835.3A CN202010242835A CN111363763A CN 111363763 A CN111363763 A CN 111363763A CN 202010242835 A CN202010242835 A CN 202010242835A CN 111363763 A CN111363763 A CN 111363763A
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杨治庆
韦阳道
万逸
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Hainan University
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Abstract

本发明可用于分析检测及基因编辑领域，能够相应的提供一种RNA激活Cas14a“附带切割”活性即可被激活成为非特异性的ssDNase的方法，解决RNA不能激活Cas14a酶的附带切割活性的问题，拓展Cas14a酶由DNA到RNA的应用范围。

Description

一种RNA激活Cas14a酶附带切割效应的方法
技术领域
本发明涉及一种RNA激活CRISPR-Cas14a酶附带切割效应的方法，可用于分析和基因编辑领域。
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：第一类是CRISPR-Cas系统利用多蛋白复合物，第二类是CRISPR-Cas系统利用单蛋白效应物[2]。在第2类CRISPR系统中，一个具有多功能域的100-200 KDa Crispr Associated protein(Cas)蛋白在RNA引导下进行DNA或RNA底物的结合和切割。CRISPR和CRISPR关联基因(CRISPR-Cas)具有的可编程的外切酶活性使其成为有效的核酸诊断工具。基于不同的效应蛋白家族，第二类系统可以分为3大类和9个亚型，其中II型的蛋白效应器Cas9与tracrRNA、引导crRNA组成效应复合物切割靶DNA[3]；V型系统的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]；VI型效应器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]。
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“附带切割”活性即可被激活成为非特异性的ssDNase的方法，对分析和基因编辑领域具有重要意义；
[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现存激活CRISPR-Cas酶附带切割效应存在的问题，提供一种RNA激活CRISPR-Cas14a酶附带切割效应的方法。
为实现上述目的，本发明采用的技术方案为：
一种RNA激活CRISPR-Cas14a酶附带切割效应的方法，向含有单链引导RNA(sgRNA)-Cas14a复合体的溶液中加入靶链RNA，即可激活Cas14a的附带切割效应；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含有与靶链RNA互补配对的间隔序列，浓度为0-100mM；所述Cas14a的浓度为0-100 mM；所述靶链RNA长度为5-10000 bp；所述sgRNA的序列为：TTC ACT GAT AAA GTG GAG AAC CGC TTC ACCAAA AGC TGT CCC TTA GGG GAT TAG AAC TTG AGT GAA GGT GGG CTG CTT GCA TCA GCCTAA TGT CGA GAA GTG CTT TCT TCG GAA AGT AAC CCT CGA AAC AAA TTC ATT TGA AAGAAT AAG GAA TGC AAC+间隔序列；
所述间隔序列为含有与靶链RNA互补5-40 bp长度的序列。
附图说明
图1 RNA激活Cas14a酶附带切割活性荧光信号图。
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的sgRNA与500 nM的Cas14a在25mM 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 UACCUUACACCGCUUGCGAA
实施例2：
RNA激活Cas14a附带切割性能：分别将100 nM的RNA1和RNA2加入实施例1中孵育好的sgRNA-Cas14溶液中，随后加入单链DNA荧光猝灭探针FQ,在96孔板中进行荧光检测，以荧光信号变化实现对激活Cas14a的附带切割活性验证，以水代替RNA1和RNA2作为空白对照组（参见图1）；
RNA1序列：UUCGCAAGCGGUGUAAGGUA
RNA2序列：AUGCGCAGGCGUGUUCGCAAGCGGUGUAAGGUAGCAGGCGUGUCG
FQ序列：Fam-TTTTTTTTTTTTTTTT-BHQ
由图1可知，含有靶链RNA1和RNA2的实验组荧光信号剧烈增强，而空白对照组荧光信号没有变化，这表明，RNA可以激活Cas14酶附带切割效应成为非特异性的ssDNse，使得FQ探针断裂，荧光信号增强。

Claims (6)

1.一种RNA激活CRISPR-Cas14a酶附带切割效应的方法，其特征在于：向含有单链引导RNA(sgRNA)-Cas14a复合体的溶液中加入靶链RNA，即可激活Cas14a的附带切割效应。
2.按权利要求1所述的RNA激活CRISPR-Cas14a酶附带切割效应的方法，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RNA激活CRISPR-Cas14a酶附带切割效应的方法，其特征在于：所述浓度为0-100mM；所述Cas14a的浓度为0-100 mM。
4.按照权利1所述的RNA激活CRISPR-Cas14a酶附带切割效应的方法，其特征在于：所述靶链RNA长度为5-10000 bp。
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sgRNA的序列为TTCACTGATAAAGTGGAGAACCGCTTCACCAAAAGCTGTCCCTTAGGGGATTAGAACTTGAGTGAAGGTGGGCTGCTTGCATCAGCCTAATGTCGAGAAGTGCTTTCTTCGGAAAGTAACCCTCGAAACAAATTCATTTgaaaGAATGAAGGAATGCAAC+间隔序列。
6.按照权利5所述sgRNA序列，其特征在于：间隔序列为含有与靶链RNA互补5-40 bp长度的序列。
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CN112680536A (zh) * 2021-02-02 2021-04-20 海南大学 一种基于crispr-cas12f1检测病原微生物RNA的方法
CN113621686A (zh) * 2021-08-11 2021-11-09 海南微氪生物科技股份有限公司 一种适用于多种病原微生物的检测方法
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
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WO2022240262A1 (ko) * 2021-05-14 2022-11-17 주식회사 진코어 Rna-guided nuclease를 이용한 lca10 치료용 조성물 및 치료방법

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WO2022075808A1 (ko) * 2020-10-08 2022-04-14 주식회사 진코어 Crispr/cas12f1 시스템 효율화를 위한 u-rich tail을 포함하는 엔지니어링 된 가이드 rna 및 그 용도
WO2022075816A1 (ko) * 2020-10-08 2022-04-14 주식회사 진코어 Crispr/cas12f1(cas14a1) 시스템 효율화를 위한 엔지니어링 된 가이드 rna 및 이의 용도
WO2022075813A1 (ko) * 2020-10-08 2022-04-14 주식회사 진코어 Crispr/cas12f1 시스템 효율화를 위한 엔지니어링 된 가이드 rna 및 그 용도
KR20220047502A (ko) * 2020-10-08 2022-04-18 주식회사 진코어 CRISPR/Cas12f1(Cas14a1) system 효율화를 위한 engineered guide RNA 및 이의 용도
KR102638799B1 (ko) 2020-10-08 2024-02-22 주식회사 진코어 CRISPR/Cas12f1(Cas14a1) system 효율화를 위한 engineered guide RNA 및 이의 용도
CN112176035A (zh) * 2020-10-14 2021-01-05 杭州优思达生物技术有限公司 一种新型crispr核酸检测方法及应用
WO2022077687A1 (zh) * 2020-10-14 2022-04-21 杭州优思达生物技术有限公司 一种新型crispr核酸检测方法及应用
CN112680536A (zh) * 2021-02-02 2021-04-20 海南大学 一种基于crispr-cas12f1检测病原微生物RNA的方法
WO2022240262A1 (ko) * 2021-05-14 2022-11-17 주식회사 진코어 Rna-guided nuclease를 이용한 lca10 치료용 조성물 및 치료방법
CN113621686A (zh) * 2021-08-11 2021-11-09 海南微氪生物科技股份有限公司 一种适用于多种病原微生物的检测方法
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
CN114196752B (zh) * 2021-12-08 2023-08-08 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

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