Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025008
The tumor suppressor p53, an indispensable nuclear transcription factor, plays a central role in orchestrating cellular responses when DNA damage occurs. In this study, we demonstrate that in the initial phases of DNA double-strand break (DSB) repair, p53 is rapidly recruited to sites of damage and the surrounding chromatin, where it enhances DSB repair efficiency. This enhancement occurs through the modulation of chromatin dynamics and the promotion of a more relaxed chromatin configuration, a process influenced by p53 in response to DSB-inducing factors such as etoposide, ultraviolet radiation, and nucleases. These results underscore the pivotal function of p53 as a rapid responder to DSBs, delineating a significant departure from its traditionally recognized role as a downstream transcriptional regulator in DNA damage repair processes. This study emphasizes that the direct engagement of p53 in DNA repair through chromatin structure regulation extends beyond its established involvement in UV irradiation-induced nucleotide excision repair (NER), demonstrating analogous mechanistic attributes in the context of DSB repair. This newly illuminated perspective enhances our understanding of the multifaceted roles of p53 in genome stability and integrity.
Acta Biochimica et Biophysica Sinica•2024•DOI: 10.3724/abbs.2024073
Chikungunya virus (CHIKV) is a neglected arthropod-borne and anthropogenic alphavirus. Over the past two decades, the CHIKV distribution has undergone significant changes worldwide, from the original tropics and subtropics regions to temperate regions, which has attracted global attention. However, the interactions between CHIKV and its host remain insufficiently understood, which dampens the need for the development of an anti-CHIKV strategy. In this study, on the basis of the optimal overexpression of non-structural protein 4 (nsP4), we explore host interactions of CHIKV nsP4 using mass spectrometry-based protein-protein interaction approaches. The results reveal that some cellular proteins that interact with nsP4 are enriched in the ubiquitin-proteasome pathway. Specifically, the scaffold protein receptor for activated C kinase 1 (RACK1) is identified as a novel host interactor and regulator of CHIKV nsP4. The inhibition of the interaction between RACK1 and nsP4 by harringtonolide results in the reduction of nsP4, which is caused by the promotion of degradation but not the inhibition of nsP4 translation. Furthermore, the decrease in nsP4 triggered by the RACK1 inhibitor can be reversed by the proteasome inhibitor MG132, suggesting that RACK1 can protect nsP4 from degradation through the ubiquitin-proteasome pathway. This study reveals a novel mechanism by which the host factor RACK1 regulates CHIKV nsP4, which could be a potential target for developing drugs against CHIKV.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025044
Pigs, as crucial economic livestock species, possess remarkable reproductive traits and thus play a highly significant role in promoting the progress of the livestock industry. With the advent and application of CRISPR/Cas9 technology, researchers have explored genetic editing techniques to increase swine reproductive performance, flavour profiles, and nutritional attributes. Additionally, with respect to anatomy, physiology, immunology, and genomics as well as other traits, pigs exhibit remarkable similarities to humans. Genetically edited pigs play crucial roles in human disease models, xenotransplantation, breed improvement, vaccine development, and drug assessment. Common methods deployed in the preparation of genetically edited pigs include somatic cell nuclear transfer (SCNT), microinjection and sperm-mediated approaches. For example, Shen et al. [1] successfully generated P53-knockout Diannan miniature pigs using transcription activator-like effector nucleases combined with SCNT, offering a valuable resource for preclinical oncology research. In 2019, Chen et al. [2] employed microinjection to deliver Cas9 messenger ribonucleic acid (mRNA) and single guide ribonucleic acid (sgRNA) into the cytoplasm of fertilized eggs. These authors successfully obtained both the albinism phenotype and the combined phenotype of albinism and immunodeficiency in Tibetan miniature pigs. More recently, Tenihara et al. [3] introduced the CRISPR/Cas9 protein into fertilized porcine eggs via electroporation, enabling a simple, micromanipulation-free approach for generating gene-edited pigs. Among these methods, SCNT has gained extensive interest among researchers because of its reliability. An essential aspect of SCNT is the preparation of embryonic fibroblasts to serve as donor cells. Previously, the CRISPR/Cas9 plasmid editing system served as the predominant technique to generate genetically edited embryonic fibroblasts (Figure 1A) [4]. This approach, which is distinguished by its relative simplicity, high stability, and low cost, was formerly widely utilized in the production of gene-edited pigs. However, plasmid editing is associated with several notable limitations. First, it introduces resistance genes, posing risks of inaccurate gene editing, drug resistance and biosafety concerns. Second, during the CRISPR/Cas9 editing process, there is a possibility of ongoing editing due to deoxyribonucleic acid (DNA) integration. This continuous editing can increase the likelihood of off-target effects, random mutations, and interference with DNA repair mechanisms. Third, the acquisition of positive cell lines via the plasmid editing system typically demands an extended period of in vitro cultivation (lasting 3–4 weeks), which increases the risk of apoptosis and chromosomal aberrations. Consequently, plasmid-based transfection is now largely supplanted by ribonucleoprotein (RNP) systems for gene editing. RNP systems bypass plasmids, delivering the Cas9 protein and sgRNA directly into cells, reducing off-target effects and cytotoxicity [5]. In 2022, Xu et al. [6] developed the reporter RNA-enriched dual-sgRNA CRISPR/Cas9 ribonucleoprotein (RE-DSRNP) method, a transgene-free approach using CRISPR/Cas9 RNPs enriched with ATTO550-tracrRNA (IDT, Iowa, USA) as a fluorescent RNA probe (Figure 1B). This method reduced the time needed to generate donor cells from 3-4 weeks to one week, resulting in high-efficiency WIP1 gene knockouts and the production of pigs with male reproductive disorders. However, owing to genetic diversity, not all target genes achieve 95% editing efficiency, as demonstrated by the RE-DSRNP method, with some falling below 90%. For example, DOCK8, which belongs to the DOCK family, is an atypical guanine nucleotide exchange factor that plays a crucial role in immune responses. DOCK8 deficiency syndrome, a rare hereditary disorder, often leads to combined immunodeficiency and is characterized by elevated serum immunoglobulin E levels, increased eosinophil
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025044
The generation of genetically edited pigs via somatic cell nuclear transfer (SCNT) has historically relied on plasmid-based CRISPR/Cas9 systems, which introduce resistance genes, risk off-target effects from prolonged editing, and require 3–4 weeks of in vitro selection. This study presents a transgene-free, rapid strategy using the IRE-DSRNP method to edit monoclonal porcine fetal fibroblasts. Three IgA-knockout cell lines were obtained with large deletions in the CH1-CH3 region: 1044 bp (heterozygous), 1043 bp (homozygous), and 1039 bp (homozygous). These cells were pooled and used as donor nuclei for SCNT. From 880 fresh oocytes, 660 mature oocytes were selected, 500 underwent enucleation and nuclear transfer, yielding 400 fused cells; 300 embryos were transplanted into a surrogate sow. Pregnancy was confirmed at 28 days, and after 143 days of gestation, six F0 piglets were born. Genotyping revealed two heterozygotes (4010#, 4015#) and four homozygous knockouts, with two piglets per genotype (1044, 1043, 1039 bp deletions). This approach eliminates plasmid integration, reduces off-target risks, and shortens the timeline for producing IgA-deficient Bama pig models, offering a robust platform for disease modeling and xenotransplantation research.