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bioRxiv · 10.1101/2025.04.15.646138

Super Recombinator (SuRe): An in vivo recombination system for scalable and efficient transgene assembly at a single genomic locus

Luo, J.·Huang, C.·Taylor, C. A.·Li, J.·Woo, S. J.·Yu, C.·Shen, K.·Schnitzer, M. J.
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Abstract

The capacity to engineer organisms with multiple transgenic components is crucial to synthetic biology and basic biology research. For the former field, transgenic organisms allow the creation of novel biological functions; for the latter, such organisms provide potent means of dissecting complex biological pathways. However, the size limitations of a single transgenesis event and challenges associated with the assembly of multiple DNA fragments hinder the efficient integration of multiple transgenes. To overcome these hurdles, here we introduce a building block for synthetic design termed an integrated genetic array (IGA), which incorporates all genetic components into a single locus to prevent their separation during genetic manipulations. Since the natural recombination rate for genes located in the same locus is near zero, to construct IGAs we developed the Super Recombinator (SuRe) system, which uses CRISPR/Cas9, alone or in combination with site-specific serine recombinases, for in vivo transgene recombination at a single genomic locus. SuRe effectively doubles the number of elements assembled in each recombination round, exponentially accelerating IGA construction. By preventing the separation of transgenic elements, SuRe greatly reduces screening burdens, as validated here through studies of Drosophila melanogaster and Caenorhabditis elegans. To optimize SuRe, we compared CRISPR/Cas9-induced homology-directed recombination to site-specific recombination using various serine recombinases. Optimized versions of SuRe achieved efficiency and fidelity values near their theoretical maxima and allowed the generation of recombinant products up to 4.2 Mbp in size in Drosophila. Using SuRe, we created fruit flies with 12 transgenic elements for fluorescence voltage imaging of neural activity in precisely defined cell-types. Mathematical modeling of the scalability of SuRe to large transgene assemblies showed that integration times and gene assembly workloads respectively scale logarithmically and linearly with the number of transgenes, both major improvements over conventional approaches. Overall, SuRe enables the efficient integration of multiple genes at individual loci, up to the chromosomal scale.

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Luo, J., Huang, C., Taylor, C. A., Li, J., Woo, S. J., Yu, C., Shen, K., Schnitzer, M. J.. 2025-04-15. Super Recombinator (SuRe): An in vivo recombination system for scalable and efficient transgene assembly at a single genomic locus. https://doi.org/10.1101/2025.04.15.646138

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