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bioRxiv · 10.1101/2025.05.27.656450

Characterization of recombinase-based genetic parts and circuits using nanopore sequencing

Greco, F. V.·Cameron, S. K.·Joshi, S. H.-N.·Guiziou, S.·Brophy, J. A. N.·Grierson, C. S.·Gorochowski, T. E.
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Abstract

Recombinases are versatile enzymes able to perform the precise insertion, deletion, and rearrangement of DNA and can act as a foundation for programmable genetic logic and memory. Fundamental to their use are accurate measurements of function. However, these are often laborious, time-consuming, and costly to collect. To address this, we developed a semi-automated workflow that combines low-cost liquid handling robotics, multiplexed long-read nanopore sequencing, and a supporting computational analysis tool to enable the high-throughput and detailed characterization of recombinase parts and circuits when used in a variety of contexts and organisms. Our approach overcomes the limitations of typically used fluorescence-based assays and is able to monitor temporal dynamics, observe structural changes at a nucleotide resolution, and unravel the internal workings of complex multi-state circuits. The ability to scale-up and automate genetic circuit characterization is an essential step towards more rigorous biological metrology that can support the construction of predictive models for efficiently engineering biology.

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Greco, F. V., Cameron, S. K., Joshi, S. H.-N., Guiziou, S., Brophy, J. A. N., Grierson, C. S., Gorochowski, T. E.. 2025-05-28. Characterization of recombinase-based genetic parts and circuits using nanopore sequencing. https://doi.org/10.1101/2025.05.27.656450

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