ObjectiveTo evaluate the expression level of histone deacetylase 9 (HDAC9) in lung squamous cell carcinoma (LUSC) tissues, to analyze its correlations with clinicopathological characteristics and prognosis of LUSC patients, and to explore the effect it exerts on the proliferation of LUSC cells.MethodsThe expression level of HDAC9 was detected by immunohistochemistry staining (IHC), and its correlations with clinicopathological characteristics were analyzed by χ2 test. Survival analysis was performed using Kaplan-Meier method. Univariate and multivariate Cox proportional hazards model were employed to analyze independent predictors for overall survival (OS) of LUSC patients. CRISPR/dCas9 activation system was used to activate the transcription of HDAC9 gene in LUSC cell line EBC-1. CCK8 cell proliferation assay and colony formation test were performed to investigate the effect that transcriptional activation of HDAC9 exerts on the proliferation of LUSC cells.ResultsOf the 129 LUSC patients, 39 (30.2%) were in the HDAC9 low expression group and 90 (69.8%) were in the HDAC9 high expression group. The OS of the patients with HDAC9 high expression was shorter than that of patients with HDAC9 low expression (P=0.032). The expression level of HDAC9 was associated with tumor grade (P=0.035), primary tumor size (P=0.041), and lymph node metastasis (P=0.013). The expression level of HDAC9 (P=0.023), tumor grade (P=0.003), primary tumor size (P=0.003), and lymph node metastasis (P=0.002) were independent predictors for OS of LUSC patients. Transcriptional activation of HDAC9 promoted colony formation of LUSC cells and cell proliferating curves showed that LUSC cells with HDAC9 transcriptional activation proliferated faster than non-targeting cells (F=52.7, P=0.002).ConclusionLUSC patients with HDAC9 high expression have poorer prognosis than HDAC9 low expression ones. The expression level of HDAC9 is associated with tumor grade, primary tumor size, and lymph node metastasis, and is identified as an independent predictor for prognosis of LUSC. Transcriptional activation of HDAC9 promotes cell proliferation in LUSC. These results suggest that HDAC9 may serve as a promising biomarker for prognosis in LUSC.
Inherited retinal diseases (IRDs) are the major cause of refractory blinding eye diseases, and gene replacement therapy has already made preliminary progress in the treatment of IRDs. For IRDs that cannot be treated by gene replacement therapy, gene editing provides an alternative therapeutic method. Strategies like disruption of pathogenic variants with or without gene augmentation therapy and precise repair of pathogenic variants can be applied for IRDs with various inheritance patterns and pathogenic variants. In animal models of retinitis pigmentosa, Usher syndrome, Leber congenital amaurosis, cone rod cell dystrophy, and other disorders, CRISPR/Cas9, base editing, and prime editing showed the potential to edit pathogenic variations in vivo, indicating a promising future for gene editing therapy of IRDs.
As the most effective and popular gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR) technology has produced a revolution in biological fundamental research, medicine and biotechnology. In this review, we describe the history of the CRISPR-CRISPR-associated protein (Cas) systems, the tools of CRISPR-Cas9, CRISPR-FnCas9/RCas9, CRISPR-Cas13 and CRISPR-Cas12a, and then some comments we need to think about.
Consensus reporting items for studies in primary care (CRISP) is a newly developed measurement tool developed abroad to standardize primary health care research, so as to improve the quality of reporting and enhance the applicability, comprehensiveness, transparency and operability of reporting. The report contains 24 Entries that follow the Introduction, Methods, Results, Discussion (IMRaD) format, and is primarily concerned with describing the research team, the patients, the study subjects, the health status, the clinical experience, the health care team, the interventions, the study interventions, and the findings in the PHC study / implementation of results, etc. This article introduces and interprets the reporting guidelines to help researchers better understand and apply this statement to improve the quality of reporting in primary health care research
ObjectiveTo compare four different transfection reagents for transfection efficiency of rat heart myoblast cells H9C2, to choose the optimal transfection method.MethodsThe plasmids of enhanced green fluorescent protein (EGFP) gene were transfected as exogenous genes to H9C2 cells from four different transfection regents including FuGENE HD, DNA-In CRISPR, Lipofectamine 3000 and Lipofectamine 2000. Fluorescence intensity was measured by fluorescence microscopy and fluorescence microplate reader to evaluate transfection efficiency. The effects of four transfection reagents on cell viability were measured by Cell Counting Kit-8 (CCK-8) reagents.ResultsTransfection efficiency of Lipofectamine 3000 was the highest (>50%), while that of DNA-In CRISPR was the lowest (<1%). The cytotoxicity of Lipofectamine 3000 was the lowest in the four transfection reagents and the cell viability was 94.55% after 48-hour transfection.ConclusionTransfection regent Lipofectamine 3000 has the relatively high transfection efficiency as well as the lowest cytotoxicity, which is more suitable for use in H9C2 cells by transfection.
In recent years, with the rapid development of gene editing technologies, research on the application of the clustered regularly interspaced short palindromic repeats (CRISPR) system in inherited retinal diseases (IRD) has become increasingly in-depth. Many IRD, such as retinitis pigmentosa, Leber congenital amaurosis, and Stargardt disease, are characterized by clearly defined pathogenic gene mutations, making them ideal targets for gene therapy. Owing to its high efficiency, strong specificity, and programmability, CRISPR technology offers a novel approach for the precise treatment of these conditions. This review summarizes recent progress in the application of CRISPR in IRD therapy, with a focus on target gene selection, optimization of editing tools and delivery systems, in vitro and in vivo validation, and early clinical investigations. In addition, current challenges, including off target effects, immune responses, and limitations in editing and delivery efficiency, are discussed. With continuous improvements in editing platforms and delivery strategies, CRISPR holds great promise for personalized treatment of IRD and may further accelerate the clinical application of precision medicine in ophthalmology.
CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system. The artificial virus could efficiently load with the CRISPR-Cas9 system, accelerate the endosomal escape, and promote the penetration into the nucleus without additional nuclear-localization signal, thus enabling targeted gene disruption. Notably, the artificial virus is more efficient than SuperFect, Lipofectamine 2000, and Lipofectamine 3000. When loaded with a CRISPR-Cas9 plasmid, it induced higher targeted gene disruption efficacy than that of Lipofectamine 3000. Furthermore, the artificial virus effectively targets the ovarian cancer via dual-receptor mediated endocytosis and had minimum side effects. When loaded with the Cas9-hMTH1 system targeting MTH1 gene, RRPHC showed effective disruption of MTH1 in vivo. This strategy could be adapted for delivering CRISPR-Cas9 plasmid or other functional nucleic acids in vivo.
As pigs are similar to humans in anatomy, physiology and pathology, nutrition metabolism and disease characteristics, genetically modified pigs are already used for the studies of disease mechanism, pathology and toxicology and the evaluation of drugs. But the production of large modified animals is difficult, cumbersome, time-consuming and costly. With the breakthrough of gene editing technology, clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated 9( Cas9)(CRISPR/Cas9) technology has greatly improved the mutation efficiency, reduced the cost and simplified the steps, and promoted the widespread application of genetically modified pigs. In this paper, the production methods of genetically modified pigs and the research progress of genetically modified pigs by CRISPR/Cas9 in the medical field were reviewed.
The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA in vitro to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.
