To achieve a reliable and persistent expression over time, the transgene should be precisely integrated into the genome safe harbor (GSH) loci. Little attention has been paid to find the safe harbor loci of the chicken (Gallus gallus domesticus) genome. Identification and characterization of GSH loci that allow the persistent and reliable expression of knocked-in genes could be a major area of interest within the field of transgenic technology and central to the development of bioreactor transgenic livestock.
Randomly integrated transgenes might be encountered position effects and epigenetic silencing, so unstable phenotypes as well as unreliable and unpredictable expression of knocked-in transgene could be occurred. In contrast to random gene insertion, site-specific gene targeting provides a specific strategy that exploits homologous recombination to insert a transgene of interest into a pre-determined locus, specifically.
In this study, using bioinformatics, gene expression atlas, and Hi-C data, the GSH region between drg1 and eif4enif1 genes was predicted in the chicken genome. Contrary to the previous time-consuming and expensive methods, here we introduce a fast and easy-to-use pipeline allowing the prediction of orthologue GSH loci in all organisms, especially in chicken. Then, the standard design of targeting and gRNA/Cas9 vectors is explained in detail.