CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

Numerous genetic variants located in autophagy-related genes have been identified for association with various cancer risks, but the biological mechanisms underlying these associations remain largely unknown. Here we investigated their regulatory activity with a parallel reporter gene assay system in breast cancer cells and identified multiple regulatory SNP sites, including rs10514231. It was located in the second intron of ATG10 and showed gene regulatory activity in most breast cancer cells we used. Mechanistically, the T allele of rs10514231 led to ATP6AP1L downregulation by decreasing the binding affinity of TCF7L2. Overexpression of the ATP6AP1L gene in cancer cells diminished cell proliferation, migration, and invasion. Notably, ATP6AP1L downregulation correlated with breast cancer risk and with poor prognosis in patients. These results provide a plausible mechanism behind the association of rs10514231 with breast cancer risk and will be important for more effective therapeutic target identification for precision medicine.

Functional characterization of cancer risk-associated single nucleotide polymorphism (SNP) identified by genome-wide association studies (GWAS) has become a big challenge. To identify the regulatory risk SNPs that can lead to transcriptional misregulation, we performed parallel reporter gene assays with both alleles of 213 prostate cancer risk-associated GWAS SNPs in 22Rv1 cells. We disclosed 32 regulatory SNPs that exhibited different regulatory activities with two alleles. For one of the regulatory SNPs, rs684232, we found that the variation altered chromatin binding of transcription factor FOXA1 on the DNA region and led to aberrant gene expression of VPS53, FAM57A, and GEMIN4, which play vital roles in prostate cancer malignancy. Our findings reveal the roles and underlying mechanism of rs684232 in prostate cancer progression and hold great promise in benefiting prostate cancer patients with prognostic prediction and target therapies.

A biotin-labelling in situ hybridization technique was first used to physically map two single copy genes, cdc2 and prh1, in maize. These two genes are metabolically interrelated genes. The full-length cDNA clones cdc2ZmA and ZmPPI of genes cdc2 and prh1 were adopted as the probes. They are 1.3 and 1.6 kb in size, respectively. Clone cdc2ZmA was physically mapped on the long arm of chromosomes 4, 8, and 9. The percent distances from centromere to detection site were 57.9 +/- 2.7, 28.4 +/- 1.5, and 88.2 +/- 3.3. The detection rate was 19.2%. Clone ZmPPI was physically mapped on the long arm of chromosomes 4, 6, and 8. The percent distances were 53.6 +/- 1.2, 60.8 +/- 2.9 and 17.1 +/- 1.6. The detection ratio was 18.5%. The technique of chromosome ISH and the relationship between the location and function of these two genes have been discussed.

The causal SNPs leading to increased cancer predisposition mainly function as gene regulatory elements, the evaluation of which largely rely on the parallel reporter gene assay system. However, the common DNA barcode-based parallel reporter gene assay systems are troubled with tag bias, mainly due to the tag nucleotide composition. Here we describe a versatile dinucleotide-tag reporter system (DiR) that enables parallel analysis of regulatory elements with minimized bias based on the next-generation sequencing. The DiR system is also more robust than the classical luciferase assay method, especially in the investigation of moderate-level regulatory elements. We applied DiR-seq assay in the functional evaluation of the prostate cancer risk SNPs in prostate cancer cell lines and disclosed 2, and 6 regulatory SNPs in PC-3 and LNCaP cells. The DiR system has great potential to advance the functional study of risk SNPs that have associations with polygenic diseases.