Analytical Chemistry

Beibei Zhang has completed his Master’s degree at Henan Agricultural University. He is a Doctor of the State Key Laboratory of Bioelectronics, Southeast University, a double first-rate university with world first-rate disciplines. He has published several papers in reputed journals with impact factor more than 15.

This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing Cas9/sgRNAs-Associated Reverse PCR. This technique detects target DNA in three steps: cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; ligating the cleaved DNA with DNA ligase and; 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 verified by this study is ready for the clinical HPV detection.