Structure-specific DNA recombination sites: Design, validation, and machine learning-based refinement.

Abstract

Recombination systems are widely used as bioengineering tools, but their sites have to be highly similar to a consensus sequence or to each other. To develop a recombination system free of these constraints, we turned toward <i>attC</i> sites from the bacterial integron system: single-stranded DNA hairpins specifically recombined by the integrase. Here, we present an algorithm that generates synthetic <i>attC</i> sites with conserved structural features and minimal sequence-level constraints. We demonstrate that all generated sites are functional, their recombination efficiency can reach 60%, and they can be embedded into protein coding sequences. To improve recombination of less efficient sites, we applied large-scale mutagenesis and library enrichment coupled to next-generation sequencing and machine learning. Our results validated the efficiency of this approach and allowed us to refine synthetic <i>attC</i> design principles. They can be embedded into virtually any sequence and constitute a unique example of a structure-specific DNA recombination system.