Vol. 12, Issue 12 (2023)

Author(s):
Mitek Tarang, Subodh Kumar, Arvind Sonwane, Amrutham Sandeep, Insha Zafir and Shahista Sarin Lodhi

Abstract:
RNA-guided recombinase (RGR) is a fusion protein composed of nuclease-null or dead Cas9 (dCas9) and ‘hyperactivated’ recombinase. RNA-guided recombinases platforms are potentially valuable tools for basic research and genome editing. The platform recombines a typical target site that comprises of a degenerate recombinase site, flanked by a 5 to 6-base pair spacer sequences and this whole central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites which is followed by protospacer adjacent motifs (PAMs). Here we have constructed a detailed map of target sites for RNA-guided recombinase platforms based on hyperactivated recombinase Beta throughout the buffalo genome. Firstly, Chromosome wise sequence data of buffalo genome was retrieved from NCBI. Then we designed search pattern for recombinase Beta with spacer length six. RGR target sites were located using this search pattern through Fuzznuc program of Emboss package. The result reveals 426 potential RGR target sites in buffalo genome for recombinase Beta with spacer length six. Out of these 207 RGR target sites lie in genic region and 263 RGR target sites in intergenic region. This work represent a step towards bringing genetic improvement in buffalo and livestock breeding as these RGR target sites provide potential of being utilized for specific genomic integration, deletion or inversion.