Myogenic Potential in Labeo rohita Dorsal Muscle Cell Line and Development of CRISPR-Cas9 Construct for Myostatin...

Labeo rohita, commonly known as rohu, is a freshwater fish from the Cyprinidae family. Myostatin (MSTN), a member of the Transforming Growth Factor-β (TGF-β) superfamily, is a negative regulator of skeletal muscle growth. CRISPR-Cas9 has progressively enhanced genome-editing efficacy by targeting various sites in the genome. The dorsal muscle cell line developed from L. rohita, named LRDM, was maintained in L-15 media containing 5% FBS and cultured for up to 35 passages. Cells were cryopreserved at different passages, achieving a 50-70% revival efficiency. PAX7 and MYOD protein expression were assessed in the LRDM to confirm the myogenic capacity at the 10th, 20th, and 30th passages. PAX7 expression was prominent in early passages, while MYOD expression became more noticeable in later passages. Guide RNAs were designed to target the L. rohita MSTN gene. One-step digestion and ligation were performed by ligating the annealed oligos to the T7cas9sgRNA2 vector. The isolated plasmid containing the target sequence was validated by using Sanger sequencing. The gRNA plasmid and pT3TS-nls-zCas9-nls (pT3 Cas9) vector were digested by using the BamHI and XbaI restriction enzymes. The expected size bands were observed at ∼2541 and ∼7332 bp. In vitro transcription of the gRNA and synthesis of pT3 Cas9 were carried out using MEGAshortscript T7 and mMESSAGE mMACHINE T3 kits, respectively. Establishing a myostatin-knockout fish muscle cell line could serve as a valuable model for studying the molecular mechanisms underlying muscle development, including cultivated fish meat production, which will be pivotal for the scientific and technological advancements needed to advance cellular aquaculture.