[发明专利]一种环形RNA敲低载体快速构建试剂盒及其应用

权利要求书

1.一种试剂盒在快速构建环形RNA敲低载体中的应用，其特征在于，包含LinearizedCasRx Vector、DNA Ligase Mix、10×Ligase Buffer、Nuclease-free Water、10μM的U6Primer、gRNA-NC Vector共6种成分；所述Linearized CasRx Vector选自LinearizedCasRx Vector，From pCasRx-L1；Linearized CasRx Vector，From pCasRxN-L1；Linearized CasRx Vector，From pCasRx-A1和Linearized CasRx Vector，From pCasRxN-A1中的一个或多个；所述Linearized CasRx Vector的序列组成为TTTT-U6 terminator-CMV promoter-CasRx-Flag-EGFP-IRES-PuroR- WPRE-backbone-U6 promoter-CasRxDR30-AAAC；所述Linearized CasRx Vector，From pCasRx-L1和 Linearized CasRxVector，From pCasRxN-L1为慢病毒载体骨架；所述Linearized CasRx Vector，FrompCasRx-A1和Linearized CasRx Vector，From pCasRxN-A1为ssAAV载体骨架；所述Linearized CasRx Vector，From pCasRxN-L1和 Linearized CasRx Vector，FrompCasRxN-A1中CasRx的5’和3’末端分别添加有核定位信号NLS序列；所述Linearized CasRxVector，From pCasRx-L1的核苷酸序列如SEQ ID NO.1；所述Linearized CasRx Vector，From pCasRxN-L1的核苷酸序列如SEQ ID NO.2；所述Linearized CasRx Vector，FrompCasRx-A1的核苷酸序列如SEQ ID NO.3；所述Linearized CasRx Vector，From pCasRxN-A1的核苷酸序列如SEQ ID NO.4；所述Linearized CasRx Vector，From pCasRx-L1为pCasRx-L0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRx Vector，FrompCasRxN-L1为pCasRxN-L0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRxVector，From pCasRx-A1为pCasRx-A0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRx Vector，From pCasRxN-A1为pCasRxN-A0经BpiI双酶切线性化后回收制备而成；所述gRNA-NC Vector选自gRNA-NC Vector，pCasRx-L1、gRNA-NC Vector，pCasRxN-L1、gRNA-NC Vector，pCasRx-A1和gRNA-NC Vector，pCasRxN-A1中的一个或多个；所述Linearized CasRx Vector的浓度为10ng/μL；所述试剂盒的连接反应为5μL体系，且gRNA(50μM)退火双链产物与Linearized CasRx Vector的比例为1：2（V/V）；所述DNA LigaseMix包含500U/μL的T4 DNA ligase、pH为7.5，20mM的Tris-HCl、50mM KCl、1mM DTT、0.1mMEDTA和50%甘油；所述10×Ligase Buffer包含pH为7.8，400mM Tris-HCl、100mM MgCl2、100mM DTT、5mM ATP和25% PEG 4000；所述10μM的U6 Primer为合成的U6引物，核苷酸序列信息如SEQ ID NO.5所示，并经PAGE纯化，使用时Nuclease-free Water稀释；所述gRNA-NCVector的序列组成为backbone-U6 promoter-CasRx DR30-non-targeting gRNA-U6terminator-CMV promoter-CasRx-Flag-EGFP-IRES-PuroR-WPRE；所述gRNA-NC Vector，pCasRx- L1和gRNA-NC Vector，pCasRxN-L1为慢病毒载体骨架；所述gRNA-NC Vector，pCasRx-A1和gRNA-NC Vector，pCasRxN-A1为ssAAV载体骨架；所述gRNA-NC Vector,pCasRxN-L1和gRNA-NC Vector，pCasRxN-A1中CasRx的5’和3’末端分别添加有核定位信号NLS序列；所述gRNA-NC Vector，pCasRx-L1的核苷酸序列如SEQ ID NO.6；所述gRNA-NCVector，pCasRxN-L1的核苷酸序列如SEQ ID NO.7；所述gRNA-NC Vector，pCasRx-A1的核苷酸序列如SEQ ID NO.8；所述gRNA-NC Vector，pCasRxN-A1的核苷酸序列如SEQ ID NO.9。