Bioinformatic identification and annotation of naturally occurring hyperactivated beta recombinase recognition sites in the goat genome

Introduction

Precise modification of genomic DNA has transformed animal biotechnology, enabling the development of gene-edited livestock with improved production traits, enhanced disease resistance, and applications in agricultural, pharmaceutical, and biomedical research. Goats, valued for their production of meat, milk, fibre, and other by-products, have also emerged as important animal models owing to their moderate body size, short reproductive cycle, and high milk yield. Consequently, genome engineering technologies have been widely applied in goats to investigate gene function, improve economically important traits, and develop models for biomedical research and gene therapy. The development of precise and efficient technologies for targeted genome modification remains a central goal of modern biotechnology. Among the available genome-editing approaches, programmable nucleases have emerged as the predominant platforms owing to their ability to introduce site-specific genetic alterations [1,2] . Among the available genome-editing tools, site-specific recombinases (SSRs) enable precise DNA rearrangements by catalysing the cleavage, strand exchange, and re-ligation of doublestranded DNA without relying on endogenous DNA repair pathways or generating insertion/deletion (indel) mutations [3,4,5] . Site-specific recombinases are DNA-modifying enzymes that catalyse precise genetic recombination at defined recognition sequences. Naturally occurring recombinases mediate targeted DNA rearrangements through specific interactions with their cognate recognition sites, enabling the deletion, insertion, inversion, or exchange of DNA segments. These enzymes have become valuable tools for genome engineering because they facilitate predictable and highly specific modifications without relying on endogenous DNA repair pathways [6] . Site-specific recombinases have become important components of genome engineering strategies owing to their ability to mediate precise and predictable DNA rearrangements. In sheep and goats, recombinase-based genome manipulation has been successfully achieved using systems such as Cre recombinase, Flp recombinase, and PhiC31 integrase [7] . Consequently, extensive efforts have focused on engineering recombinases with enhanced catalytic activity and expanded target specificity. Advances in directed evolution, high-throughput screening, and protein engineering have accelerated the development of recombinase variants with modified DNA-binding specificities [8,9] . In parallel, detailed characterization of recombinase substrate specificity has provided valuable insights for the rational design and evolution of enzymes capable of recognizing user-defined genomic targets [10,11] . Although only the Gin (β)-based RGR platform has been established to date, several additional hyperactivated recombinases have been evaluated in zinc-finger recombinase (ZFR) platforms [2,12] . Four additional Gin variants (α, γ, δ, ε, and ζ) have also been characterized 2 and ZFR platforms based on β and Sin recombinases have demonstrated functional activity 12 . Target sites for RGR platforms based on recombinases other than Gin (β) can be identified by replacing the central 20-bp pseudo-recognition sequence with the corresponding recombinase-specific sequence 13 . The continued development of hyperactivated recombinases is expected to expand the targeting range and versatility of recombinase-based genome-editing systems. Accordingly, the present study aimed to generate a genomewide map of target sites for the hyperactivated Beta recombinase with a spacer length of five base pairs and to identify genes associated with these target sites across the goat genome, thereby facilitating future applications of RNAguided recombinase-mediated genome editing.

Material and Method 2.1 Identification of hyperactivated Beta recombinase recognition sites with a 5-bp spacer in the goat genome

The reference genome assembly of goat (Capra hircus) (ARS1.2; RefSeq assembly accession GCF_001704415.2) and the corresponding genome annotation (GFF3) file were obtained from the NCBI RefSeq database. Genome-wide identification of hyperactivated Beta recombinase recognition sites with five base spacer was performed using a custom Python pipeline developed with the Biopython and `regex` libraries. Previously, it has been reported [12] that the core sequence for hyperactivated recombinase Beta as: NNNV NNR GT WW AC YNN BNNN where N=A, T, C, or G; V=A, C, or G; B=T, C, or G; R=A or G; Y=T or C; W=A or T. Therefore, in present study search pattern was designed according to the experimentally determined DNA recognition specificity of the hyperactivated Beta recombinase described by Sirk et al., (2014). The recognition sequence consisted of fixed terminal nucleotides (5′-CC and GG-3′), variable left and right binding regions, and a conserved 5-bp spacer. Degenerate nucleotide positions were represented using regular-expression character classes to permit all nucleotide combinations experimentally tolerated at each position while preserving the conserved bases of the recognition motif. Genome-wide scanning was performed on both the forward and reverse DNA strands. For reverse-strand analysis, the reverse-complement sequence of each chromosome was generated, and the genomic coordinates of reverse-strand matches were converted back to the corresponding coordinates of the reference genome. Each identified recognition site was recorded with its chromosome accession, genomic start and end positions, strand orientation, and complete recognition sequence.

Chromosomal distribution of Beta recombinase recognition sites

The chromosomal distribution of Beta recombinase recognition sites was determined by counting the number of identified sites on each chromosome. In addition, the distribution of recognition sites across genomic features, including CDS, exons, gene-associated regions, and intergenic regions, was summarized to evaluate their genomic context. All computational analyses were performed using custom Python scripts executed under Python 3.13 on a Windows operating system.

Genomic features annotation of identified Beta recombinase recognition sites

The identified Beta recombinase recognition sites were subsequently annotated using the corresponding goat genome GFF3 annotation. Each recognition site was compared with annotated genomic features to determine whether it overlapped an annotated gene or was located within an intergenic region. For gene-associated sites, the corresponding gene symbol and strand orientation were recorded. Featurelevel annotation was further performed by comparing each recognition site with annotated coding sequences (CDS), exons, and gene regions. Recognition sites overlapping CDS or exon features were classified accordingly, whereas sites overlapping annotated gene regions without intersecting CDS or exon features were classified as gene-associated (intronic/gene region). Sites showing no overlap with annotated genes were classified as intergenic.

Functional enrichment analysis

Genes containing Beta recombinase recognition sites were subjected to functional enrichment analysis using Gene Ontology (GO) and KEGG pathway analyses through g. Enrichment analyses included GO Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) categories. Significantly enriched GO terms and KEGG pathways were identified using the default multiple-testing correction implemented in g, and terms with adjusted P values < 0.05 were considered statistically significant.

Results

Genome-wide identification of hyperactivated Beta recombinase recognition sites

A genome-wide computational survey identified 914 naturally occurring recognition sites for the hyperactivated Beta recombinase in the Capra hircus (ARS1.2) reference genome. Screening of both forward and reverse DNA strands revealed that all identified recognition sites represented 914 unique genomic loci, indicating the absence of complementary duplicate matches. The identified sites were distributed throughout the goat genome, including autosomes, sex chromosomes, and unlocalized scaffolds, demonstrating the widespread A5SAoccurrence of sequences compatible with the hyperactivated Beta recombinase recognition motif.

Chromosome-wise distribution of Beta recombinase recognition sites

The identified Beta recombinase recognition sites were unevenly distributed across the goat chromosomes (Figure 1). Recognition sites were detected on nearly all assembled chromosomes, although their abundance varied considerably among chromosomes. In general, larger chromosomes contained a greater number of recognition sites than smaller chromosomes, reflecting the genome-wide distribution of the Beta recombinase recognition motif.

Distribution of recombinase recognition sites across genomic features

Annotation against the RefSeq genome annotation identified 414 recognition sites located within annotated genes, corresponding to 201 unique genes, while the remaining 500 sites were located in intergenic regions (Figure 2). Geneassociated recognition sites were distributed among proteincoding genes involved in diverse biological functions, whereas intergenic sites represented the largest genomic category, indicating that naturally occurring Beta recombinase recognition motifs are widely dispersed throughout both coding and non-coding regions of the goat genome.

Functional enrichment analysis

Functional enrichment analysis of the 201 unique genes containing Beta recombinase recognition sites revealed significant enrichment across the three GO categories: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) (Table 1 and Figure 3). In the Biological Process (BP) category, the most significantly enriched terms were biological regulation (adjusted P = 5.93 × 10⁻⁴; 112 genes) and regulation of biological process (adjusted P = 2.52 × 10⁻³; 108 genes), indicating that a large proportion of the identified genes participate in diverse regulatory mechanisms. Additional significantly enriched processes included platelet morphogenesis, platelet formation, and synapse organization, suggesting the involvement of these genes in developmental and cellular signaling pathways. Within the Molecular Function (MF) category, protein binding was the most significantly enriched term (adjusted P = 7.31 × 10⁻⁵; 93 genes), demonstrating that many candidate genes encode proteins involved in molecular interactions. Other enriched molecular functions included enzyme activator activity, enzyme regulator activity, GTPase regulator activity, nucleoside-triphosphatase regulator activity, and GTPase activator activity, indicating that the identified genes are associated with the regulation of enzyme activity and intracellular signaling processes. For the Cellular Component (CC) category, cell junction showed the strongest enrichment (adjusted P = 2.97 × 10⁻⁶; 33 genes). Other significantly enriched cellular components included glutamatergic synapse, cell periphery, and synapse, suggesting that many genes are localized to membraneassociated and cell communication structures. The TEAD-YAP complex was also significantly enriched, although represented by only two genes. Overall, the enrichment analysis indicates that genes containing or located near the identified recombinase recognition sites are predominantly involved in biological regulation, protein interaction, enzyme modulation, intracellular signaling, and cell junction-and synapseassociated functions. These findings suggest that the identified genomic target sites are associated with genes involved in fundamental cellular and regulatory processes

Discussion

The present study provides the first comprehensive genomewide survey of naturally occurring recognition sites for the hyperactivated Beta recombinase in the Capra hircus reference genome. Using a computational motif-search strategy based on the experimentally characterized five-base spacer recognition sequence, a total of 914 unique Beta recombinase recognition sites were identified across the goat genome. The widespread distribution of these sites demonstrates that sequences compatible with the engineered Beta recombinase are naturally present throughout the genome, thereby expanding the repertoire of potential target loci for recombinase-mediated genome engineering in goats. Unlike programmable nuclease-based genome-editing systems, which rely on the induction of DNA double-strand breaks and endogenous DNA repair pathways, site-specific recombinases catalyze precise DNA rearrangements through conservative recombination between defined recognition sites, enabling predictable genomic modifications with minimal dependence on host repair mechanisms. Consequently, engineered serine recombinases have emerged as attractive alternatives for precise genome engineering applications [2,12,13] . The large number of naturally occurring Beta recognition sites identified in the present study indicates that the goat genome contains numerous candidate loci that could potentially be exploited for targeted recombinase-mediated DNA integration, excision, or inversion. Genomic annotation revealed that 414 recognition sites were located within annotated genes representing 201 unique genes, whereas the remaining recognition sites were located in intergenic regions. The predominance of intergenic sites is advantageous for future genome engineering applications because intergenic regions are generally less likely to disrupt protein-coding sequences or regulatory elements, making them attractive candidates for targeted insertion of transgenes or other genetic modifications. Nevertheless, the presence of numerous gene-associated recognition sites also provides opportunities for precise manipulation of endogenous genes involved in economically important traits or disease resistance. Functional enrichment analysis demonstrated that genes containing Beta recombinase recognition sites were significantly enriched for Gene Ontology terms associated with biological regulation, enzyme activator activity, enzyme regulator activity, GTPase regulator activity, nucleosidetriphosphatase regulator activity, and GTPase activator activity, platelet morphogenesis, platelet formation, and synapse organization. These enriched biological processes suggest that Beta recognition sites are widely distributed among genes involved in fundamental cellular and developmental functions rather than being restricted to a particular functional category. Similar observations have been reported for genome-wide surveys of engineered recombinase recognition sequences in livestock genomes, where candidate target sites were broadly distributed across functionally diverse genomic regions [12,13] . The enrichment of signaling-related pathways supports the GO enrichment results and indicates that genes harboring Beta recombinase recognition sites participate in complex biological networks. Importantly, these findings do not imply preferential recombinase activity within these pathways but rather reflect the functional characteristics of genes naturally containing sequences compatible with the Beta recombinase recognition motif. The present study was based on computational prediction of naturally occurring recognition sites using the goat reference genome and experimentally defined Beta recombinase sequence specificity. Consequently, the identified recognition sites represent candidate genomic targets that require experimental validation to determine recombination efficiency, target accessibility, chromatin context, and editing specificity in vivo. Future studies combining computational prediction with cell-based recombination assays and genome engineering experiments will be essential to validate these candidate loci and assess their suitability for practical applications. Nevertheless, several challenges remain, including improving recombination efficiency, minimizing off-target activity, understanding the influence of post-translational modifications on enzyme stability and function [14,15] and developing safe and efficient delivery strategies for therapeutic applications [16,17,18,19] . Addressing these challenges will be essential for realizing the full potential of engineered site-specific recombinases as versatile tools for genome engineering and gene therapy.

Conclusion

This study establishes the first genome-wide catalogue of naturally occurring hyperactivated Beta recombinase recognition sites in the goat genome. The widespread genomic distribution of these sites and their association with genes involved in diverse biological processes provide a valuable resource for future recombinase-mediated genome engineering. These findings expand the available genomic information for engineered serine recombinases and may facilitate the development of precise genome-editing strategies for functional genomics, livestock genetic improvement, and translational biotechnology.

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