FUNCTIONAL ENRICHMENT ANALYSIS OF GENES ASSOCIATED WITH NATURALLY OCCURRING HYPERACTIVATED BETA RECOMBINASE RECOGNITION SITES WITH A SIX-BASE SPACER IN THE CAPRA HIRCUS GENOME

Abstract

Hyperactivated serine recombinases are promising genome engineering tools because they mediate precise DNA recombination without inducing double-strand breaks. However, the genomic distribution of endogenous hyperactivated Beta recombinase recognition sites with six base spacers in goat (Capra hircus) genome has not been systematically investigated. A genome-wide computational search was performed using the Capra hircus ARS1.2 reference genome and a degenerate recognition motif for the hyperactivated Beta recombinase with a 6-bp spacer. Target sites identified on both DNA strands were merged, duplicate loci were removed, and unique sites were annotated using the reference GFF3 file. Functional enrichment of associated genes was assessed using Gene Ontology (GO) and KEGG pathway analyses. A total of 509 unique Beta recombinase recognition sites were identified across the goat genome. Most sites were located in intergenic (52.7%) and intronic (40.7%) regions, while a smaller proportion occurred within CDS (3.1%), promoters (2.2%), and exons (1.4%). Functional enrichment analysis revealed significant GO terms related to cellular regulation, signaling, and small GTPase-mediated signal transduction. GO Molecular Function analysis highlighted protein binding and GTPase regulator activity, whereas GO Cellular Component analysis showed enrichment of cytosolic and cytoplasmic components. KEGG analysis identified axon guidance as the significantly enriched pathway. This study presents the first genome-wide catalogue of naturally occurring hyperactivated Beta recombinase recognition sites with six base spacer in the goat genome. These findings provide a valuable resource for recombinase-mediated genome engineering and future precision genetic improvement in goats.