Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique
Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique
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使用新型 Cas9/sgRNA 相关反向 PCR (CARP) 技术检测目标 DNA
DOI：
10.1007/s00216-018-0873-5
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发表时间：
2018-05-01
影响因子：
4.3
通讯作者：
Wang, Jinke
中科院分区：
文献类型：
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作者：
Zhang, Beibei;Wang, Qiao;Wang, Jinke
本研究开发了一种基于Cas9核酸酶检测靶DNA的新方法，命名为CARP，代表CRISPR-或Cas9/sgRNAs-associated reverse PCR。该技术通过三步检测目标DNA:(1)用Cas9复合物与目标DNA特异性的一对sgrna切割检测到的DNA样本；(2)用DNA连接酶连接被切割的DNA；(3)用PCR扩增靶DNA。在连接步骤中，将cas9切割的靶DNA连接成分子内环状或分子间连接的线性DNA。在PCR步骤中，用一对反向引物扩增连接的DNA。通过检测9种不同人乳头瘤病毒（HPV）亚型的HPV16和HPV18 L1基因，验证了该技术。该技术还检测了两种hpv阳性宫颈癌细胞（HeLa和SiHa）基因组DNA中两种高危hpv （HPV16和HPV18）的L1和E6-E7基因，而hpv阴性宫颈癌细胞C-33a的基因组DNA中未检测到L1和E6-E7基因。通过进行这些概念验证实验，本研究提供了一种新的基于crispr的DNA检测和分型方法。特别是本研究开发的CARP方法已经可以用于临床HPV检测，并得到了最终临床样本检测的支持。
This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing CRISPR- or Cas9/sgRNAs-associated reverse PCR. This technique detects target DNA in three steps: (1) cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; (2) ligating the cleaved DNA with DNA ligase; (3) amplifying target DNA with PCR. In the ligation step, the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA. In the PCR step, the ligated DNA was amplified with a pair of reverse primers. The technique was verified by detecting HPV16 and HPV18 L1 genes in nine different human papillomavirus (HPV) subtypes. The technique also detected the L1 and E6-E7 genes of two high-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells (HeLa and SiHa), in which no L1 and E6-E7 genes were detected in the HPV-negative cervical carcinoma cell, C-33a. By performing these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method. Especially, the CARP method developed by this study is ready for the clinical HPV detection, which was supported by the final clinical sample detection.