KRYPTONINT has delivered multiple advances that push the boundaries of current knowledge in microbial genetics, integron biology, and biotechnology:
- Development of SeqDelTA, the first method to successfully delete highly stabilized SCIs such as the Superintegron of V. cholerae, overcoming the challenge posed by 19 toxin-antitoxin systems. This enables the study of SCIs in their native context and is protected by international patents (ES2969666B2, ES2970040B2, PCT/ES2024/070711).
- Erasing the Superintegron without altering host physiology, revealing that SCIs are genetically and functionally isolated from the core genome. This contradicts long-held assumptions and positions SCIs as modular, accessory units. This insight was published in Nucleic Acids Research (2024).
- Discovery of gene-less cassettes acting as internal promoters, changing our view of transcriptional regulation within long arrays. These elements modulate expression beyond the proximity to the main promoter, with consequences for co-selection and resistance dynamics. This is complemented by our finding on polar effects and the new translation-driven polarity model of expression of integrons. Together, both findings shift the paradigm of expression of the integron (Nature Communications, 2024; Nucleic Acids Research, 2024).
- Identification of integrase expression heterogeneity, a novel finding showing that integrase is expressed in a subset of cells even without stress. This phenotypic heterogeneity leads to genotypic diversification, unveiling a mechanism of bacterial pre-adaptation with strong evolutionary implications (manuscript in preparation, expected 2025).
- Discovery that mobile integrons encode phage defence systems, revealing a second major function for integrons beyond antibiotic resistance. This breakthrough has major implications for the future of phage therapy and was published in Science (2025).
- Creation of the I3C tool, a cassette capture platform that is agnostic to cassette sequence or function. It allows direct capture of cassettes from DNA samples and has led to the discovery of new antibiotic and phage resistance genes. This tool is protected by additional patents (ES2991744A1, PCT pending), with commercial interest from private equity funds and plans for future exploitation via an ERC Proof of Concept.
- Cross-disciplinary integration of microbiology, molecular genetics, single-cell analysis and synthetic biology, with high throughput and resolution.
By the end of the project, KRYPTONINT has not only resolved key fundamental questions about integron biology but has also delivered innovative tools and conceptual frameworks that redefine the field. The outputs of the project have high potential for translation into diagnostics, synthetic biology, and antimicrobial resistance surveillance.
