Characterizing and engineering bridge recombinases for large-scale genome engineering

• Applicant: Dr. Gabriel Mello da Cunha Longo, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 568545862

Harnessing the naturally occurring enzymes to programmably modify DNA sequences has revolutionized biomedical science, paving the way for new genetic medicines by directly correcting pathogenic mutations. However, the genetic heterogeneity of nearly all genetic diseases necessitates a tailored gene-editing approach for each mutation, making it impractical from cost, time, and scale perspectives to develop new treatments for each patient. One possible solution is to insert functional copies of entire genes at programmed regions of the genome, allowing correction of genetic diseases in a mutation-agnostic fashion. However, the precise and efficient insertion of large DNA sequences remains a preeminent challenge in the genome editing field. Recently, IS110 and IS1111 families of mobile genetic elements have been characterized, leading to the discovery of autonomous systems composed of a recombinase and a non-coding bridge RNA (bRNA) that mediates targeted DNA recombination. These elements enable site-specific large-scale DNA editing and given their easy of reprograming to new target sites, are promising candidates to overcome challenges of current technologies. However, bridge recombination activity is not effective in human cells, motivating experiments to improve efficiencies. Here, I propose to develop more effective bridge recombinases that are highly functional in human cells by exploring the natural diversity of IS110/1111 elements to identify, characterize, and further engineer bridge recombinases for programmable insertion of large DNA sequences in human genomes. The aims of this project are: (1) screen and define the programmability rules of hundreds of bridge recombinases while engineering the previously characterized IS621 recombinase (eIS621s) and (2) investigate the specificity of uncharacterized IS elements and eIS621s, assessing their potential as gene editors for inserting corrective transgenes. To achieve these goals, I will develop a high-throughput next generation sequencing-based assay to screen active IS elements in E. coli, and will devise a directed evolution approach to engineer IS621 for higher recombination activity in human cells. Next, I will characterize the safety landscape of the enzymes by investigating off-target recombination in human cells, seeking to ensure high specificity integration at the on-target site. Finally, I will validate bridge recombinase-mediated large sequence insertion in an engineered cell line carrying the most common genetic mutation found to cause phenylketonuria (PKU), a common genetic liver disorder caused by mutations in the PAH gene. By investigating the untapped diversity of IS elements and engineering bridge recombinases, this project aims to harness the potential of this elegant mechanism for the next generation of genome editors.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Dr. Benjamin Kleinstiver