Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off‐target effects, including the cleavage of unintended targets, limits the practical applications of Cas9‐mediated genome editing. In this study, to minimize the off‐target effects of this technology, we utilized D10A‐Cas9 nickase to generate myostatin‐knockout (MSTN KO) chickens via primordial germ cells. D10A‐Cas9 nickase (Cas9n)‐mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild‐type chickens. Our results demonstrate that the D10A‐Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome‐edited chickens in practical industrialization and as animal models of human diseases.

Chicken as a food source is one of the most widespread domestic animals, and it has been used extensively as a research model. The clustered regularly interspaced short palindromic repeats (CRISPR)‐CRISPR‐associated protein 9 (Cas9) system is the most efficient and reliable tool for precise genome‐targeted modification and has generated considerable excitement for industrial applications, as well as biologic science. Unlike in mammals, germline‐transmittable primordial germ cells (PGCs) in chicken were used as an alternative strategy for the production of genetically altered chickens. Here, by combining the CRISPR‐Cas9 platform and germ cell‐mediated germline transmission, we generated G ₀ /G ₁ switch gene 2 ( G0S2 )knockout (KO)chickens, and G0S2 null KO chickens showed a dramatic reduction of abdominal fat deposition without affecting other economic traits. Additionally, G0S2 null KO chickens had altered fatty acid compositions in their blood and abdominal fat compared with wild‐type chickens under normal dietary conditions. The global mRNA sequencing data showed that G0S2 disruption in chickens would activate the adipose tissue‐specific peroxisomal oxidation pathway, and enoyl‐coenzyme A (CoA), hydratase/3‐hydroxyacyl CoA dehydrogenase might be a target molecule in metabolic homeostasis in the chicken adipose tissue. Our results demonstrate that the CRISPR‐Cas9 system with chicken PGCs can facilitate the production of specific genome‐edited chickens for practical applications, as well as basic research.—Park, T. S., Park, J., Lee, J. H., Park, J.‐W., Park, B.‐C., Disruption of G0/G1 switch gene 2 (G0S2) reduced abdominal fat deposition and altered fatty acid composition in chicken. FASEB J. 33, 1188–1198 (2019). www.fasebj.org

Genes, RNAs, and proteins play important roles during germline development. However, the functions of non-coding RNAs (ncRNAs) on germline development remain unclear in avian species. Recent high-throughput techniques have identified several classes of ncRNAs, including micro RNAs (miRNAs), small-interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs). These ncRNAs are functionally important in the genome, however, the identification and annotation of ncRNAs in a genome is challenging. The aim of this study was to identify different types of small ncRNAs particularly piRNAs, and the role of piRNA pathway genes in the protection of chicken primordial germ cells (PGCs).

Monocytes/macrophages, which are found in a variety of organs, maintain tissue homeostasis at a steady state and act as the first line of defence during pathogen-induced inflammation in the host. Most monocyte/macrophage lineage studies in chickens have been largely performed using cell lines, while few studies using primary cells have been conducted. In the present study, the phenotypic and functional characteristics of splenic monocyte/macrophage lineage cells during steady state and inflammatory conditions were examined. Splenic monocyte/macrophage lineage cells could be identified as MRC1^loMHCII^hiand MRC1^hiMHCII^locells based on their surface expression of MRC1 and MHCII. In the steady state, MRC1^loMHCII^hicells were more frequently found among MRC1⁺cells. MRC1^loMHCII^hicells expressed a higher number of antigen-presenting molecules (MHCII, MHCI, and CD80) than MRC1^hiMHCII^locells. In contrast, MRC1^hiMHCII^locells showed better phagocytic and CCR5-dependent migratory properties than MRC1^loMHCII^hicells. Furthermore, MRC1^hiMHCII^locells infiltrated the spleen in vivo and then became MRC1^loMHCII^hicells. During lipopolysaccharide (LPS)-induced inflammatory conditions that were produced via intraperitoneal (i.p.) injection, the proportion and absolute number of MRC1^hiMHCII^locells were increased in the spleen. Uniquely, inflammation induced the downregulation of MHCII expression in MRC1^hiMHCII^locells. The major source of inflammatory cytokines (IL-1β, IL-6, and IL-12) was MRC1^loMHCII^hicells. Furthermore, MRC1^hiMHCII^locells showed greater bactericidal activity than MRC1^loMHCII^hicells during LPS-induced inflammation. Collectively, these results suggest that two subsets of monocyte/macrophage lineage cells exist in the chicken spleen that have functional differences.

Objective: In the poultry industry, the most important economic traits are meat quality and carcass yield. Thus, many studies were conducted to investigate the regulatory pathways during muscle differentiation. To gain insight of muscle differentiation mechanism during growth period, we identified and validated calcium-related genes which were highly expressed during muscle differentiation through mRNA sequencing analysis. Methods: We conducted next-generation-sequencing (NGS) analysis of mRNA from undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods). Subsequently, we obtained calcium related genes related to muscle differentiation process and examined the expression patterns by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results: Through RNA sequencing analysis, we found that the transcription levels of six genes (troponin C1, slow skeletal and cardiac type [TNNC1], myosin light chain 1 [MYL1], MYL3, phospholamban [PLN], caveolin 3 [CAV3], and calsequestrin 2 [CASQ2]) particularly related to calcium regulation were gradually increased according to days of myotube differentiation. Subsequently, we validated the expression patterns of calcium-related genes in quail myoblasts. These results indicated that TNNC1, MYL1, MYL3, PLN, CAV3, CASQ2 responded to differentiation and growth performance in quail muscle. Conclusion: These results indicated that calcium regulation might play a critical role in muscle differentiation. Thus, these findings suggest that further studies would be warranted to investigate the role of calcium ion in muscle differentiation and could provide a useful biomarker for muscle differentiation and growth.