Precise genome editing is essential for both basic and translational research. The recently developed CRISPR/Cas9 system can specifically cleave a designated site of target gene to create a DNA double-strand break, which triggers cellular DNA repair mechanism of either inaccurate non-homologous end joining, or site-specific homologous recombination. Unfortunately, homology-directed repair (HDR) is challenging due to its very low efficiency. Herein, we focused on improving the efficiency of HDR using a combination of CRISPR/Cas9, eGFP, DNA ligase IV inhibitor SCR7, and single-stranded oligodeoxynucleotides (ssODN) in human cancer cells. When Cas9, gRNA and eGFP were assembled into a co-expression vector, the disruption rate more than doubled following GFP-positive cell sorting in transfected cells compared to those unsorted cells. Using ssODNs as templates, SCR7 treatment increased targeted insertion efficiency threefold in transfected cells compared to those without SCR7 treatment. Moreover, this combinatorial approach greatly improved the efficiency of HDR and targeted gene mutation correction at both the GFP-silent mutation and the β-catenin Ser45 deletion mutation cells. The data of this study suggests that a combination of co-expression vector, ssODN, and ligase IV inhibitor can markedly improve the CRISPR/Cas9-directed gene editing, which should have significant application in targeted gene editing and genetic disease therapy.

Hypermethylation of specific gene promoters is an important mechanism of carcinogenesis. A high frequency of promoter methylation of PAX1 and ZNF582 genes has been detected in cervical cancer. In the present study, we investigated the methylation status of PAX1 and ZNF582 genes in esophageal squamous cell carcinoma (ESCC) tissues. Tumor and paracancerous tissues were obtained from 14 ESCC patients. Genomic DNA was extracted from both tumor and paracancerous tissues, and the concentration of DNA were determined. DNA methylation analysis of PAX1 and ZNF582 genes was carried out using quantitative methylation-specific PCR. To assess the diagnostic performance of the two methylated genes for cancer detection, receiver operating characteristic (ROC) curves were generated. Sensitivities and specificities were tested at cut-offs obtained from the ROC curves. The methylation levels of both PAX1 and ZNF582 genes were significantly higher in tumor tissues compared to non-tumor paracancerous tissues. The methylation rates of PAX1 and ZNF582 in ESCC tumor and paracancerous tissues were 100% and 21.4% (p = 0.006), 85.7% and 0% (p < 0.001), respectively. The sensitivities and specificities of PAX1 and ZNF582 methylation for the detection of cancer were 100% and 85.7%, and 78.6% and 100%, respectively. The DNA methylation levels and frequencies of PAX1 and ZNF582 genes were markedly higher in ESCC tumor tissues compared to those in paracancerous tissues. Moreover, the conclusions were verified by using The Cancer Genome Atlas (TCGA) datasets. DNA methylation status of these two genes showed a relatively good sensitivity and specificity for the detection of ESCC tumors. This data suggests that DNA methylation testing holds a great promise for ESCC screening and warrants further prospective population-based studies.