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CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method

Qiao Wang, Beibei Zhang, Xinhui Xu, Feifei Long, Jinke Wang
doi: https://doi.org/10.1101/236588
Qiao Wang
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China
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Beibei Zhang
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China
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Xinhui Xu
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China
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Feifei Long
Nanjing Foreign Language School, Nanjing 210096, China
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Jinke Wang
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China
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  • For correspondence: wangjinke@seu.edu.cn
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Abstract

This study develops a new method for detecting and typing target DNA based on Cas9 nuclease, which was named as ctPCR, representing Cas9/sgRNA- or CRISPR-typing PCR. The technique can detect and discriminate target DNA easily, rapidly, specifically, and sensitively. This technique detects target DNA in three steps: (1) amplifying target DNA with PCR by using a pair of universal primers (PCR1); (2) treating PCR1 products with a process referred to as CAT, representing Cas9 cutting, A tailing and T adaptor ligation; (3) amplifying the CAT-treated DNA with PCR by using a pair of general-specific primers (gs-primers) (PCR2). The technique was verified by detecting HPV16 and HPV18 L1 gene in 13 different high-risk human papillomavirus (HPV) subtypes. The technique was also detected two high-risk HPVs (HPV16 and HPV18) in cervical carcinoma cells (HeLa and SiHa) by detecting the L1 and E6/E7 genes, respectively. In this method, PCR1 was performed to determine if the detected DNA sample contained the target DNA (such as virus infection), while PCR2 was performed to discriminate which genotypic target DNA was present in the detected DNA sample (such as virus subtypes). With these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method.

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Posted December 19, 2017.
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CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method
Qiao Wang, Beibei Zhang, Xinhui Xu, Feifei Long, Jinke Wang
bioRxiv 236588; doi: https://doi.org/10.1101/236588
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CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method
Qiao Wang, Beibei Zhang, Xinhui Xu, Feifei Long, Jinke Wang
bioRxiv 236588; doi: https://doi.org/10.1101/236588

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