Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.
Alleles ; CRISPR-Cas Systems ; DNA End-Joining Repair ; Gene Editing/methods* ; Recombinational DNA Repair

DNA End-Joining Repair ; Humans ; Immunologic Deficiency Syndromes ; genetics

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180degrees and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.
DNA End-Joining Repair ; DNA Transposable Elements ; DNA* ; Enhancer Elements, Genetic ; Hand ; Homologous Recombination ; Retroelements

Among the genotoxic drug regimens, doxorubicin (DOX) is known for its high-dose side effects in several carcinomas, including cervical cancer. This study reports on testing the combined use of a DOX genotoxic drug and SCR-7 non-homologous end joining (NHEJ) inhibitor for HeLa cells. An in vitro DNA damaging assay of DOX was performed on plasmid and genomic DNA substrate. In vitro cytotoxicity was investigated using trypan blue dye exclusion, DNA metabolizing, and propidium iodide-based flow cytometric assays. DOX (between 20–100 μM) displayed clear DNA binding and interaction, such as the shearing and smearing of plasmid and genomic DNA. DNA metabolizing assay data indicate that HeLa lysate with DOX and SCR-7 treatment exhibited better in vitro plasmid DNA stability compared with DOX treatment alone. SCR-7 augmented the effects of low-dose DOX by demonstrating enhanced cell death from 15% to 50%. The flow cytometric data also supported that the combination of SCR-7 with DOX lead to a 23% increase in propidium iodide-based HeLa staining, thus indicating enhanced death. In summary, the inhibition of NHEJ DNA repair pathway can potentiate low-dose DOX to produce appreciable cytotoxicity in HeLa cells.
Cell Death ; DNA ; DNA Damage ; DNA End-Joining Repair ; DNA Repair ; Doxorubicin* ; Drug Therapy ; Genomic Instability ; HeLa Cells* ; Humans ; In Vitro Techniques ; Plasmids ; Propidium ; Trypan Blue ; Uterine Cervical Neoplasms

Eukaryotic organisms constantly face a wide range of internal and external factors that cause damage to their DNA. Failure to accurately and efficiently repair these DNA lesions can result in genomic instability and the development of tumors (Canela et al., 2017). Among the various forms of DNA damage, DNA double-strand breaks (DSBs) are particularly harmful. Two major pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), are primarily responsible for repairing DSBs (Katsuki et al., 2020; Li and Yuan, 2021; Zhang and Gong, 2021; Xiang et al., 2023). NHEJ is an error-prone repair mechanism that simply joins the broken ends together (Blunt et al., 1995; Hartley et al., 1995). In contrast, HR is a precise repair process. It involves multiple proteins in eukaryotic cells, with the RAD51 recombinase being the key player, which is analogous to bacterial recombinase A (RecA) (Shinohara et al., 1992). The central event in HR is the formation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments that facilitate homology search and DNA strand invasion, ultimately leading to the initiation of repair synthesis (Miné et al., 2007; Hilario et al., 2009; Ma et al., 2017).
Recombinational DNA Repair ; DNA-Binding Proteins/metabolism* ; DNA Repair ; DNA Damage ; DNA

OBJECTIVE: The resistance mechanisms of tumor cells to chemotherapeutic drugs are known as the followings; the alterations in the drug transport and activation, the enhanced expression of the DNA repair and replication and the decreased apoptosis. The aim of this study is to examine a relative difference on the level of the mRNA expression of the multidrug resistance (MDR)-related and the apoptosis-associated genes between cisplastin-sensitive and cisplatin-resistant human ovarian cancer cell line. METHODS: MDR-associated genes (lrp, mdr1/p-glycoprotein, mrp) and PKC isozymes (alpha, beta1, beta2, epsilon, eta, theta), DNA mismatch repair (MMR) genes (hMLH1, hMSH2, hMSH3, hMSH6), DNA topology-related genes (topoisomerase IIalpha and beta) and apoptosis-related genes (p53, p21, mdm2, fas (Apo-1), trail (Apo-2L) were analyzed in cisplatin-sensitive ovarian cancer cell line A2780 and -resistant cell line A2780cp by complementary DNA polymerase chain reaction. RESULTS: The mdr1 and PKC eta in mRNA level were expressed in A2780cp, but not in A2780. The mRNA expressions of lrp, p21 and mdm2 were more increased in A2780cp than drug sensitive variant A2780, but not significantly correlated. In contrast mRNA expression of hMLH1, a kind of DNA MMR gene, was remarkably decreased and mRNA expression of hMSH2 was slightly decrease in A2780cp. However, the levels of mrp, topo II alpha and beta, hMSH3, hMSH6, p53, fas and trail were not affected. CONCLUSION: These results showed that mdr1/p-gp expression may be an important determinant of MDR phenotype in resistant cell line to chemotherapeutic agents, and PKC isozymes and DNA MMR genes may be responsible for cisplatin resistant in ovarian cancer.
Apoptosis* ; Cell Line* ; Cisplatin* ; DNA ; DNA Mismatch Repair ; DNA Repair ; DNA, Complementary ; Drug Resistance, Multiple* ; Humans* ; Isoenzymes ; Ovarian Neoplasms* ; Phenotype ; Polymerase Chain Reaction ; RNA, Messenger

Consensus ; DNA Repair ; Humans ; Male ; Prostatic Neoplasms/genetics* ; Recombinational DNA Repair

Microsatellite mutator phenotype (MMP) tumors were reported in a subset of gastrointestinal carcinomas. The molecular pathogenesis of MMP tumors shows defects in the DNA mismatch repair genes, and also many germline and somatic mutations were reported in the MMP tumors. However, the detection of genetic defects in the MMP tumors is very difficult, mainly because many genes are included in the DNA mismatch repair genes. This study was undertaken to determine the best strategy for detecting defects in the DNA mismatch repair genes in gastrointestinal carcinomas. One of the effective ways for detecting defects in DNA mismatch repair genes is to screen the MMP tumors and evaluate the products of DNA mismatch repair genes by performing the multiplex RT-PCR method. We have screened the MMP tumors by using 5 microsatellite markers in the 12 cancer cell lines, 120 colon carcinomas and 99 gastric carcinomas and found 6 MMP cell lines, 10 MMP colon cancers, and 9 MMP gastric carcinomas. In addition, we evaluated 6 DNA mismatch repair gene products (hMSH2, hMSH3, hMSH6, hMLH1, hPMS1 and hPMS2) by multiplex RT-PCR analysis and found decreased expression of the DNA mismatch repair genes in 5 (hMSH6 in DLD-1 and HCT-15; hMSH2 in LoVo; hMLH1 and hMSH3 in HCT-116; hMLH1 in SNU-638) out of 6 MMP cell lines. We also found a decreased expression of hMLH1 in 3 out of 10 MMP colon carcinomas, and in 6 out of 9 MMP gastric carcinomas. Our results indicate that the expression analysis of the DNA mismatch repair genes by multiplex RT-PCR method can reduce the number of genes subjected to mutational analysis and is convenient for screening the responsible DNA mismatch repair genes.
Cell Line ; Colon ; Colonic Neoplasms ; DNA Mismatch Repair* ; DNA* ; Mass Screening ; Microsatellite Repeats* ; Phenotype*

BACKGROUND/AIMS: Serrated adenoma of the colorectum is a recently proposed entity that is characterized by a saw-toothed structure of hyperplastic polyp and also the cytologic atypia of conventional adenoma. In contrast to conventional adenomas, the molecular features of serrated adenomas have been poorly studied. METHODS: The expression of beta-catenin and the DNA mismatch repair protein hMLH1, apoptosis regulating protein Bcl-2, Bax, p53 and COX-2 were analyzed in 28 serrated adenoma specimens and 28 tubular adenoma specimens. RESULTS: No differences were observed in the frequency of beta-catenin loss in the cell membrane between the serrated and tubular adenoma specimens. The frequency of hMLH1 loss was significantly higher in the serrated adenomas than in tubular adenomas (p < 0.05). The frequency of p53 overexpression was not significantly different between the serrated and tubular adenoma specimens. The frequencies of the Bax and Bcl-2 overexpressions were significantly lower in the serrated adenomas than in the tubular adenomas. The COX-2 levels were not different between the serrated and tubular adenomas. In the serrated adenoma specimens, the frequency of Bax overexpression was reduced in the older age group (> 60 years old). In the tubular adenoma specimens, the frequency of p53 overexpression was increased in the dysplastic epithelium. CONCLUSIONS: The expressions of hMLH1, Bax and Bcl-2 were decreased in the serrated adenoma than in the tubular adenoma. Our data suggest that the serrated adenoma and tubular adenoma may have different pathway in their development. However, further studies including normal mucosa, hyperplastic polyp and cancer specimens are needed.
Adenoma* ; Apoptosis ; beta Catenin* ; Cell Membrane ; Colon* ; DNA Mismatch Repair ; Epithelium ; Humans ; Mucous Membrane ; Polyps

The major risk factor for ovarian cancer (OC) is mutation of the BRCA1 or BRCA2 DNA mismatch repair genes, which occurs in approximately 10% of OC cases. Most previous studies have demonstrated that BRCA1- and BRCA2-mutated OCs are associated with better prognosis than sporadic OCs. However, information about the patterns and clinical course of the metastatic spread of BRCA-mutated OCs is limited. Herein, we describe a case of OC with a BRCA1 mutation and skin metastases in a 49-year-old patient, which to the best of our knowledge has not been reported previously.
DNA Mismatch Repair ; Humans ; Middle Aged ; Neoplasm Metastasis* ; Ovarian Neoplasms* ; Prognosis ; Risk Factors ; Skin*