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K. Rasal

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Open access Jul 2026

Design and in silico validation of donor DNA for RNA-guided recombinase-mediated knockout of mstnb gene in Labeo rohita

Myostatin b (mstnb), a key negative regulator of skeletal muscle growth, represents a promising target gene for genome editing aimed at enhancing growth performance and aquaculture productivity. The present study aimed to design, construct and in silico validate a donor DNA carrying a single-nucleotide substitution that introduces a premature stop codon in the mstnb gene of Labeo rohita for RNA-guided recombinase (RGR) platform-mediated genome editing. For this purpose the mstnb gene sequence was retrieved from the NCBI database and analysed to identify an appropriate target site within exon 1. A targeted single nucleotide substitution from guanine to thymine (G > T) was strategically planned into the donor DNA upstream of the native stop codon to convert the glycine codon (GGA) into a premature stop codon (TGA). This was achieved by identifying RGR target sites flanking the intended mutation site and designing specific primers to amplify and clone the DNA fragment. The target 600 bp DNA fragment encompassing the mutation site flanked by two RGR target sites was successfully amplified and ligated into the pJET1.2 cloning vector and confirmed through Sanger sequencing. Site-directed mutagenesis successfully introduced the intended nucleotide substitution, which was subsequently confirmed by Sanger sequencing. Computational analyses using InterPro, ColabFold, SWISS-MODEL and CYS_REC predicted that the introduced nonsense mutation would generate a truncated Mstnb protein, resulting in the loss of conserved TGF-β domains, reduced structural stability, and impaired cytokine activity. Structural modelling further revealed disruption of the C-terminal β-sheet structure, reduced stereochemical quality, altered QMEAN Z-scores, and loss of cysteine residues, collectively indicating impaired protein folding and reduced structural stability. These findings suggest that the engineered mutation is likely to abolish the functional activity of the mstnb gene, thereby providing a validated donor DNA construct for precise RGR-mediated genome editing in L. rohita. Future studies will focus on the experimental validation of the engineered donor DNA construct through RGR-mediated genome editing, followed by functional characterization in L. rohita.

Lukram Sushil Singh, Porkodi Murugesan, Gowhar Iqbal et al. · 0 citations
Open access Jul 2026

Design and transfection of CRISPR/Cas9 constructs for the myostatin gene in Labeo rohita muscle cells

Myostatin (mstn) is a negative regulator of skeletal muscle growth and is considered as an important target for enhancing aquaculture production. The present study aimed to design and validate single-guide RNAs (sgRNAs) and CRISPR/Cas9 constructs for exon 1 of the mstnb gene in Labeo rohita, and to evaluate their transfection efficiency in the L. rohita dorsal muscle (LRDM) cell line at the 10th, 20th, and 30th passages. sgRNAs were designed and cloned into the pSpCas9(BB)-2A-GFP (PX458) vector using BbsI restriction digestion and ligation. Successful insertion and correct orientation of the sgRNAs were confirmed through Sanger sequencing. LRDM cells were revived and maintained in L-15 medium supplemented with 10% fetal bovine serum. Transfection was performed at the 10th, 20th, and 30th passages. Distinct GFP-positive cells were observed at all passages for both sgRNA constructs, indicating the ability of the developed cell line to successfully express the constructs across different passages. The study successfully established CRISPR/Cas9 plasmid constructs for the mstnb gene in L. rohita and demonstrated their transfection in LRDM cell line across multiple passages. These findings provide a basis for future studies on genome editing approaches using CRISPR/Cas9 constructs in fish muscle cell lines and highlight the potential application of CRISPR/Cas9 technology for genetic engineering applications in fish muscle cells.

Gowhar Iqbal, Nevil Pinto, Darshan Pawaskar et al. · 0 citations