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bioRxiv · 10.1101/2025.09.16.676606

Integrase-On-Demand: Bioprospecting integrases for targeted genomic insertion of genetic cargo

McClain, H. M.·Lowrey, L. C.·Quinto, L. B.·Torrance, E. L.·Gagliano, T. R.·Isaacs, F. J.·Schoeniger, J. S.·Williams, K. P.·Mageeney, C. M.
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Abstract

Integrases recognize defined sites within DNA and facilitate homologous recombination-independent transfer of genetic fragments. These innate capabilities render integrases as powerful biotechnology tools that mediate efficient site-specific integration of large genetic cargos. Given that genomes often lack the integration sites recognized by frequently utilized integrases, integrase technology has largely been restricted to genetic engineering of model organisms into which attB sites can be synthetically introduced. To enable single-step site-specific integrase-mediated genome editing in a broad spectrum of prokaryotes, we have devised the Integrase-On-Demand (IOD) method. IOD systematically identifies integrases, from bacteria and archaea, that can integrate into available attB sites in any target prokaryote. Computational results show that diverse bacteria generally have multiple potentially useable native attB/integrase pairs. We confirmed the functionality of predicted integrase and attB pairs for mediating site-specific genomic integration of heterologous DNA into the genomes of Pseudomonas putida S12 and KT2440 and Synechococcus elongatus UTEX 2973 and measured efficiency of integration using suicide constructs. By eliminating the requirement to introduce non-native attB sites into the target genome, IOD may, when suitable transformation methods exist, allow facile genome integration of large constructs in non-model and possibly non-culturable bacteria. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/676606v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1442a69org.highwire.dtl.DTLVardef@3c91b1org.highwire.dtl.DTLVardef@8131f4org.highwire.dtl.DTLVardef@1f6a206_HPS_FORMAT_FIGEXP M_FIG C_FIG

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McClain, H. M., Lowrey, L. C., Quinto, L. B., Torrance, E. L., Gagliano, T. R., Isaacs, F. J., Schoeniger, J. S., Williams, K. P., Mageeney, C. M.. 2025-09-16. Integrase-On-Demand: Bioprospecting integrases for targeted genomic insertion of genetic cargo. https://doi.org/10.1101/2025.09.16.676606

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