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

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.

Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing

“…d Combination of PCR and sgRNA/Cas9 cutting followed by A tailing and T adaptor ligation for genotyping, modified from Ref. [ 58 ]. e RNA knockdown with RCas effectors (Cas13d) and splicing with catalytically inactivated dCas13d, illustrated according to Ref.…”

“…The protocol includes PCR amplification with a universal primer pair, Cas9 cutting, A tailing and T adaptor ligation. After determining if target virus DNA exists, the amplification was performed with a pair of HPV16- or HPV18-specific primers to distinguish the subtypes of HPV [ 58 ]. Moreover, mutation signal could be similarly raised after the cleavage of wildtype fragments (Fig.…”

Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods

“…These assays combine CRISPR specific cleavage and PCR amplification of target DNA, and then the results can be visualized with gel readout or qPCR ( Figure 1C). ctPCR and CARP are verified by detecting HPV16 and HPV18 L1 gene of human papillomavirus (HPV) [22][23][24]. More recently, Wang et al combine CRISPR/Cas9 with the lateral flow assay to develop CRISPR/Cas9mediated lateral flow nucleic acid assay (CASLFA) ( Figure 1D) [25].…”

“…The system successfully provides the proof-of-concept of detecting DNA methylation and Listeria monocytogenes RNA [21]. Another group developed two methods, named CRISPR-typing PCR (ctPCR) and CRISPR-associated reverse PCR (CARP) for detecting DNA [22][23][24]. These assays combine CRISPR specific cleavage and PCR amplification of target DNA, and then the results can be visualized with gel readout or qPCR ( Figure 1C).…”

Applications of CRISPR‐Cas9 in gynecological cancer research

“…The RhoA‐/‐ROCK signaling pathway can be suppressed after knockout of the Trio gene by employing CRISPR‐Cas9 in the cervical cancer cell lines, CaSki and HeLa, thereby inhibiting the migration and metastasis of cervical cancer . In addition, CRISPR‐Cas9 has been developed for the detection and typing of HPV and can be used to screen high‐risk HPV infection in cervical cancer, and the general process as shown in the Figure .…”