Temperature-Switchable Genome Editors from Extremophile-Derived Integrases

Abstract

Integrases are site-specific recombinases encoded by phages and other mobile genetic elements. They mediate DNA integration, excision, and inversion between cognate attachment (att) sites. Although integrases are powerful tools for genetic engineering and synthetic biology, most systems lack intrinsic mechanisms that limit activity after expression, creating potential for unintended recombination. We hypothesized that extremophiles could provide temperature-responsive integrases because their enzymes evolved under selective pressure to operate within the thermal ranges experienced by their hosts. To test this concept, we linked integrase-att pairs from 458,683 prokaryotic genome assemblies to curated host growth-temperature metadata. This analysis revealed temperature-associated structure among integrase clusters and established a candidate pool for testing temperature-responsive recombinases. GC content in tyrosine integrase-associated attB sites showed a modest increase in higher-temperature hosts. We developed an inversion assay using a single-copy reporter plasmid and sacB counterselection to quantify integrase activity across temperatures. Thermophile-derived integrases from Thermus thermophilus and Geobacillus stearothermophilus displayed hot-ON/cold-OFF activity profiles, whereas an integrase derived from the psychrotroph Pseudomonas cerasi showed cold-ON/hot-OFF activity. Together, these results establish host thermal niche as a guide for discovering intrinsically temperature-switchable integrases and provide a foundation for engineering thermally controlled genome editing systems. GRAPHICAL ABSTRACT Discovery and validation of temperature-switchable integrases. (A) Integrase–attachment site pairs were identified from the TIGER/Islander resource and linked to host thermal metadata from curated growth-temperature databases. Candidate systems were grouped by host thermal class, including psychrophile, psychrotroph, mesophile, thermophile, and hyperthermophile sources. (B) Selected integrase-att pairs were tested in an E. coli inversion assay in which recombination switches a reporter cassette from a sacB-sensitive state to a sucrose-resistant state. (C) Thermophile-derived integrases exhibited hot-ON/cold-OFF recombination profiles, whereas a lower-temperature-associated Pseudomonas cerasi integrase showed cold-ON/hot-OFF activity, demonstrating that host thermal niche can guide discovery of temperature-responsive recombinases.