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.

Figure 1

Cas9-sgRNA cleavage of HPV16 and HPV18 L1 genes. (A) HPV L1 plasmids and locations of sgRNA targets and universal PCR primers in the L1 and E6-E7 genes of HPV16 and HPV18. (B) Cas9-sgRNA cleavage of HPV16 L1 gene using sgRNA 16–1274 and 16–950. (C) Cas9-sgRNA cleavage of HPV18 L1 gene using sgRNA 18–1490 and 18–1274. The Cas9 protein was in complex with the sgRNAs specific to HPV16 or 18 L1 genes and used to cut the linearized HPV16 or HPV18 L1 plasmids (A). The DNAs were run with agarose gel.

Figure 2

Detection of HPV L1 gene with ctPCR by using different primers. (A) A schematic of the experimental procedures for detecting and typing HPV DNA with ctPCR by using different primers. (B) Detection of HPV16 L1 gene by ctPCR with different primers. (C) Detection of HPV18 L1 gene by ctPCR with different primers. The final PCR products were run with agarose gel. Single primer, a general primer complementary to the constant T adaptor used in ctPCR detection; Sg-primer, a pair of primers complementary to constant T adaptor and 3 nucleotides at the end of Cas9-cut product (named as general-specific primers, gs-primers).

Figure 3

Detection of HPV16 and HPV 18 genes in cervical carcinoma cells with tPCR-based ctPCR. (A) Schematic show of procedures for detecting and typing HPV DNA with tPCR-based ctPCR. (B) Detecting the HPV16 L1 and E6-E7 genes in the SiHa gDNA (200 ng) with ctPCR. (C) Detecting the HPV18 L1 and E6-E7 genes in the HeLa gDNA (200 ng) with ctPCR. The C-33a gDNA (200 ng) was detected as a negative control and a mimic detection of no DNA was used as a blank control. The final ctPCR products were run with agarose gel.

Figure 4

Detection of HPV16 and HPV 18 genes in cervical carcinoma cells with qPCR-based ctPCR. (A) Schematic show of procedures for detecting and typing HPV DNA with qPCR-based ctPCR. The reaction volumes of each step and the solutions used to the next step were shown (right). (B) Detection of the HPV16 L1 and E6-E7 genes in the three human cervical carcinoma cell lines, HeLa, SiHa and C-33a. Q-PCR1 used 200 ng gDNA of each cell line as template.

Figure 5

Detection of HPV in eight clinical samples with qPCR-based ctPCR. (A) Amplification of HPV L1 and E6-E7gene in clinical samples with qPCR1. (B) Detection of HPV16 and HPV18 L1 and E6-E7 genes in the HPV-positive clinical samples with qPCR2. S, sample. QPCR1 used the universal primers, L1-MY09/11 and E67-6F/7 R (Table 2). QPCR2 used the gs-primers (Table 2).

Figure 6

Detection of HPVs in ten clinical samples with qPCR-based ctPCR. (A) Detection of HPV L1 gene in clinical samples with qPCR1 and detection of HPV16 and HPV18 L1 genes in the HPV-positive clinical samples with qPCR2. (B) Comparison of the results of ctPCR and HC2-RDB detections. S, sample. QPCR1 used the universal primers, L1-MY09/11 (Table 2). QPCR2 used the gs-primers (Table 2). RDB, reverse dot blot. N, negative; P, positive; 16, HPV16; 18, HPV18.

Figure 7

Characterization of detection sensitivity of qPCR-based ctPCR. (A) Detection of a HPV18 L1 gene fragment (452 bp) in various dilutions with qPCR1 by using MY09/11 primers. (B) Detection of the CAT-treated qPCR1 products with qPCR2 by using gs-primers (gs-18L1-F and gs-18L1-R). (C) Schematic show of ctPCR detection operation.