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

## TL;DR

This paper introduces a new method called Integrase-On-Demand (IOD) to enable targeted gene insertion in a wide range of bacteria without needing to add special DNA sites.

## Contribution

IOD identifies native integrase-attB pairs in prokaryotes, enabling site-specific integration without synthetic attB site introduction.

## Key findings

- Diverse bacteria typically have multiple native attB sites usable for novel integrases.
- IOD successfully mediated site-specific integration in Pseudomonas putida and Synechococcus elongatus.
- Nonreplicating vectors were used to measure integration efficiency.

## Abstract

Integrases serve as powerful biotechnology tools that catalyze recombination at specific DNA sequences (att sites) and facilitate chromosomal integration of gene cargos transferred into cells. Given that genomes often lack the attB 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, within 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 sites for novel integrases. We confirmed the functionality of predicted integrase and attB pairs for mediating site-specific integration of heterologous DNA into the genomes of Pseudomonas putida S12 and KT2440 and Synechococcus elongatus UTEX 2973, measuring efficiency of integration using nonreplicating vectors. 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 nonmodel and possibly nonculturable bacteria.

Graphical Abstract