Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.

In mammals, Myostatin (MSTN) is a known negative regulator of muscle growth and development, but its role in birds is poorly understood. To investigate the molecular mechanism of MSTN on muscle growth and development in chickens, we knocked out MSTN in chicken fetal myoblasts (CFMs) and sequenced the mRNA transcriptomes. The amplicon sequencing results show that the editing efficiency of the cells was 76%. The transcriptomic results showed that 296 differentially expressed genes were generated after down-regulation of MSTN, including angiotensin I converting enzyme (ACE), extracellular fatty acid-binding protein (EXFABP) and troponin T1, slow skeletal type (TNNT1). These genes are closely associated with myoblast differentiation, muscle growth and energy metabolism. Subsequent enrichment analysis showed that DEGs of CFMs were related to MAPK, Pl3K/Akt, and STAT3 signaling pathways. The MAPK and Pl3K/Akt signaling pathways are two of the three known signaling pathways involved in the biological effects of MSTN in mammals, and the STAT3 pathway is also significantly enriched in MSTN knock out chicken leg muscles. The results of this study will help to understand the possible molecular mechanism of MSTN regulating the early differentiation of CFMs and lay a foundation for further research on the molecular mechanism of MSTN involvement in muscle growth and development.

Accurate quantification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-induced insertions and deletions (indels) is essential in chicken genome-editing studies, where low editing efficiencies and mosaicism can complicate the assessment of target-site modifications. Existing assays often involve a trade-off between affordability and the sensitivity orsequence information needed for detailed analysis. Here, we introduce IndelQuant, a cost-efficient workflow integrating sample-specific barcode PCR, pooled amplicon library construction, AdapterRemoval3-based read processing, and CRISPResso2-based indel quantification. The workflow provides graphical and tabularsummaries of editing efficiency, indel positions, indel-size distributions, and sample-level mutation profiles. We evaluated the workflow in TYR-edited DF-1 cells and genome-edited chickens. In edited chickens, IndelQuant detected tissue-specific indel signals, most below 5%, while negative controls showed background frequencies from 0 to 0.03%. These findings indicate that IndelQuant retains the sequence-level information provided by amplicon sequencing while simplifying sample processing and analysis, offering a practical approach to routine screening for low-frequency and mosaic indels in poultry genome-editing studies.