Livestock animal are important for agriculture economy and biomedical research. They are sources of Milk, meat, carcass, organic manure and other products. The development of genome editing technologies, especially CRISPR-Cas have revolutionized the generation of gene edited farm animals. In this review, we briefly introduce the CRISPR-Cas9 technology and highlight its application on livestock such as human disease modeling, disease resistant animal, and generation of hornless cattle, animal welfare and other agricultural and biomedical related traits which enhance the livestock production in order to meet the increasing demand of food worldwide. The ability to transfer sperm-producing stem cells or spermatogonial stem cells (SSCs) from a donor animal into the testes of a recipient male could have multiple applications. Production of BLG free milk in cattle provides a promising way to those who have allergy to cow milk. The knockdown of myostatin gene in different species like Sheep, Goat, Cattle and pig is very helpful in the economy of meat industry. Besides the several benefits of CRISPR-Cas9 technology, the risk factors and ethics issues related to this technology should be reconsidered before they enter into CRISPR era.

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The full open reading frame (ORF) of the buffalo beta-lactoglobulin (Bu_LALBA) gene was characterized. Results showed that Bu_LALBA ORF consisted of 429 bp long (142 aa residues) with three nucleotide variations at 111bp, 147 bp, and 291 bp but no change in amino acid sequence. The MSA showed that the Bu_LALBA was more different from the pig and human LALBA sequences. The phylogenetic tree showed that the cow, yak, and buffalo formed one cluster, while the buffalo was more closer (94%) to domestic yak (Bos grunniens). Results of ExPASy analysis showed that the Bu_LALBA protein was acidic (pI, 4.81), thermo-tolerant, and hydrophilic. However, the presence of random coil (33.80%) and α-helix (41.5%) in Bu_LALBA protein suggest that the protein was flexible and thermostable. Thirty liner motifs were identified, indicating that the Bu_LALBA act as regulatory protein. The tertiary structure of Bu_LALBA predicted by I-TASSER showed a more stabilized nature of LALBA protein. Further, the Ramachandran plot validated the 3-D structure of Bu_LALBA, which was of decent quality. The presence of four ligand-binding sites in Bu_LALBA (calcium ion, glycine, N-acetyl-L-glutamate, and N-acetylglucosamine) proposed that the LALBA binds to several fatty acids and ions. The presence of four serine, four threonine, two tyrosine residues, and six methylated lysine and five acetyl-lysine sites in Bu_LALBA indicated that the protein was involved in post-translational modification processes. IEDB analysis showed the presence of five and one epitope sites in Bu_LALBA protein for B-cell and T-cell, respectively, which suggest that this protein has certain immunological roles.

Buffalo rank second for milk production in the world and play important role in Indian economy. There are four types of caseins alpha-S1-casein, alpha-S2-casein, beta-casein and kappa-casein in milk. The major function of the casein protein in milk is to chelate colloidal calcium phosphate and serves as a major source of amino acids, calcium and phosphate. In buffalo milk all four casein proteins (alpha s1, alpha s2, beta and kappa) are encoded by four closely linked autosomal genes (CSN1S1, CSN1S2, CSN2 and CSN3 respectively) that are present on chromosome 7. Bovine CSN2 (beta-casein) gene is 8.5 kb long and contains nine exons and CSN3 (kappa-casein) is ~ 13 kb long. The aim of the study was to characterize CSN2 and CSN3 genes, In-silico analysis of beta- casein and kappa casein protein and evolutionary relationship with other species. Buffalo mammary gland tissue was collected from local slaughterhouse (New Delhi, India) and total RNA was isolated from Buffalo Mammary Epithelial Cells. The ORF region of CSN2 and CSN3 genes were amplified and sequenced for characterization. Physiochemical properties showed that both buffalo beta-casein (Bu_CSN2) and kappa-casein (Bu_CSN3) proteins are stable and hydrophobic in nature. The presence of high phosphorylated residues in both beta-casein and kappa-casein proteins residues suggested that they are involved in signal transduction processes, cell growth and metabolism. The N-glycosylation result showed that both proteins are less in foldable state. The presence of methylation and acetylation sites in both protein revealed that they are involved in different cellular process. The evolutionary analysis showed that both buffalo genes more closely to Bos grunniens (yak).