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 type 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). This method was verified by detecting HPV16 and HPV18 L1 gene in 13 different high-risk human papillomavirus (HPV) subtypes. This method was also verified by detecting the L1 and E6-E7 genes of two high-risk HPVs (HPV16 and 18) in cervical carcinoma cells and many clinical samples. 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). Based on these proof-of-concept experiments, this study provides a new CRISPR/Cas9-based DNA detection and typing method.
Highlights
DNA detection and genotyping are always of importance to the basic researches and various detecting and diagnostic applications
To further explore the applications of Cas9-single guide RNA (sgRNA), this study developed a new Cas9-based DNA detection technique, which was named as ctPCR, representing the Cas9-sgRNA- or clustered regularly interspaced short palindromic repeat (CRISPR)-typing polymerase chain reaction (PCR)
This study developed a new method for detecting target DNA based on Cas[9] nuclease, which was named as ctPCR, representing the Cas9-sgRNA-typing PCR
Summary
DNA detection and genotyping are always of importance to the basic researches and various detecting and diagnostic applications. Despite the technique still limited by the difficulty of designing specific reverse PCR primers, the great potential of CRISPR/Cas[9] application in DNA detection was preliminarily demonstrated. To further explore the applications of Cas9-sgRNA, this study developed a new Cas9-based DNA detection technique, which was named as ctPCR, representing the Cas9-sgRNA- or CRISPR-typing PCR. This study indicated that ctPCR could detect two high-risk HPVs (HPV16 and HPV18) in the human cervical carcinoma cells (HeLa and SiHa) and clinical samples by detecting both L1 and E6/E7 genes By performing these proof-of-principle detections, this study developed a new CRISPR-based PCR technique for detecting and typing DNA. The transformed E. coli was cultivated on agars with ampicillin plus chloromycetin overnight and imaged
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