A one-pot CRISPR/Cas9-typing PCR for DNA Detection and genotyping.

The CRISPR/Cas9 has high specificity to its target DNA as a gene editing tool. This characteristic makes it useful for DNA detection. Combining the advantages of CRISPR/Cas9 and PCR, this study established a novel CRISPR/Cas9-based DNA detection method, named as CRISPR/Cas9-typing PCR version 4.0 (ctPCR4.0). This method can detect target DNA in one spot with high specificity and sensitivity. In a homogenous reaction, the target DNA is first cleaved by a pair of Cas9-sgRNA complexes and thus releases two single strands with free 3' ends, allowing a pair of oligonucleotides to anneal with. The annealed oligonucleotides provide templates for DNA polymerization from the free 3' ends. A universal primer annealing site is thus produced at the end of two single strands. Then the target DNA is amplified by PCR using a universal primer. This method was first verified by accurately detecting the cloned L1 fragments of 10 genotypes of high-risk human papilloma viruses (hrHPVs). This method was then validated by detecting the L1 fragments of two highest-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells, HeLa and SiHa. Finally, this method was further validated by accurately detecting 10 hrHPVs in 30 clinical samples.