The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation.

Plesiomonas shigelloides, a Gram-negative bacterium prevalent in aquatic environments and also frequently isolated from livestock and poultry, was investigated through integrated whole-genome sequencing and functional analyses. We deciphered the pathogenic mechanisms of P. shigelloides CA-HZ1, a highly virulent strain isolated from a novel piscine host, revealing a complete genome assembly with a 3.49 Mb circular chromosome and 311 kb plasmid housing 3247 predicted protein-encoding genes. Critical genomic features included 496 virulence factors and 225 antibiotic resistance genes. Pathogenicity analysis indicated that P. shigelloides was responsible for disease outbreaks. Antimicrobial susceptibility tests showed resistance to various drugs, such as kanamycin, erythromycin, and penicillin. Histopathological examination showed significant alterations in the infected hosts. Quantitative real-time PCR (qRT-PCR) was carried out to analyze immune-related gene (IL-6, IL-1β, IL-21, STAT1, and HSP70) levels in liver and intestinal tissues, demonstrating the potent immunity triggered by P. shigelloides infection. An analysis of the liver transcriptome revealed that P. shigelloides has the potential to influence the cellular composition, molecular functions, and biological processes. Collectively, this study describes the genomic basis underlying both the pathogenic potential and hypervirulence of P. shigelloides CA-HZ1, establishing a foundational framework for investigating its broad host tropism and immune response.

Fat deposition is an important factor that affects meat production and its quality in livestock animals, including poultry. Non−coding RNAs (ncRNAs) play an important role in duck fat deposition. This study aims to systematically identify key regulatory molecules involved in fat deposition in 8−day−old Cherry Valley ducks through transcriptomic sequencing across four sample groups: intramuscular pre−adipocytes (IMP−0), intramuscular adipocytes after 4 days of induction (IMP−4), subcutaneous pre−adipocytes (SCP−0), and subcutaneous adipocytes after 4 days of induction (SCP−4). Differential expression analysis preliminarily identified several circRNAs and miRNAs differentially expressed during adipocyte differentiation, including novel_circ_000012, novel_circ_000037, novel_circ_000089, as well as miR−501−y, miR−378−y, and miR−3968−y. Further co−expression network analysis revealed that the network constructed during intramuscular adipocyte differentiation comprised 17 nodes and 39 edges, while the network constructed during subcutaneous adipocyte differentiation was larger, containing 39 nodes and 50 edges. Based on connectivity screening, we identified several key miRNAs, such as novel−m0630−5p, novel−m0485−5p, novel−m0672−5p, miR−5126−y, and miR−1408−y. Notably, this study uncovered several novel ceRNA regulatory axes during intramuscular and subcutaneous adipocyte differentiation, including novel_circ_001327/miR−141−y/Zdhhc1, novel_circ_002268/miR−2478−y/ACLY, and novel_circ_002268/miR−3963−x/ACLY. These findings provide crucial molecular insights into the specific deposition mechanisms of intramuscular versus subcutaneous fat in meat ducks, offering valuable targets for molecular breeding programs aimed at improving meat quality.