Identification of Gin-α Hyperactivated Recombinase-based RNA-Guided Recombinase Platform Target Sites in the Zebrafish Genome

Background: RNA-guided recombinase (RGR) platform is recombinase action-based genome engineering platform. It is a fusion of dead Cas9 (dCas9) and evolved recombinase with relaxed target specificity. RGR is facile, programmable and has high genome targeting capacity and a safer alternative to conventional nuclease-based methods, which carry a risk of genotoxicity. The present study was conducted to map the entire zebrafish genome for the locations of Gin-α hyperactivated recombinase-based RGR platform target sites with varying spacer lengths. Methods: RGR platform target site DNA patterns with varying spacer lengths (5/6/7-base pair) were searched in all zebrafish chromosomes using the NCBI Genome Data Viewer (GDV) genome browser. Genomic contexts of the located RGR platform target sites were further documented using NCBI Blast. Result: The study identified 262 RGR platform target sites with 5-bp spacer, 307 sites with 6-bp spacer and 326 sites with 7-bp spacer. It also analyzed the genomic context of these sites, identifying zebrafish genes located near the identified target sites. This work contributes to the development of genome editing techniques that are highly specific and safer, as well as programmable and simple and provides tools to utilize them in model organisms like zebrafish, which are commonly used in various genetic and biomedical studies.