In vitro cleavage validation assay of myostatin gene by CRISPR-Cas9 ribonucleoprotein complex: from PCR amplification to cleavage assay
摘要
The primary experimental objective of the present investigation is to study the targeted genome editing of the myostatin (MSTN) gene using ribonucleoprotein (RNP) complexes composed of Cas9 protein and single guide RNA (sgRNA). Muscle tissue from PB-1 line of chicken was used to clone the myostatin gene and sequenced using oxford nanopore technology to identify genetic variants. The sgRNAs were designed for further validation by CRISPR-Cas9 Ribonucleoprotein Complex. The Cas9 protein was incubated with sgRNA to form the RNP complex. The PCR amplification of myostatin gene produced a distinct band of 1128 bp. Analysis of the sequencing results confirmed the total size of the recombinant plasmid to be 4128 bp and the insert length of 1128 bp encoding a protein of 375 amino acids. Two single nucleotide polymorphisms (SNPs) were identified—a cytosine-to-thymine substitution at position 672 bp and a thymine-to-guanine substitution at position 699 bp of the coding region of the gene. Both the SNPs were synonymous mutations that did not alter the amino acid sequence. Incubation of PCR products with the assembled RNP complex resulted in three distinct DNA bands of 1128 bp, 764 bp, and 364 bp for sgRNA1 and 1128 bp, 835 bp and 293 bp for sgRNA2. In control samples, a band at approximately 1128 bp was observed. This pattern indicates specific cleavage by the RNP complex and our assay successfully identified two functional sgRNAs for MSTN, providing validated tools for subsequent genome editing efforts in the Indian PB-1 chicken line. This article aligns with SDG 2 (Zero Hunger) of the UN Agenda for Sustainable Development.