Recombinase-based genome editing can install kilobase-scale DNA sequences into mammalian genomes but depends on large double-stranded DNA (dsDNA) donors, which can evoke toxic innate immune responses. In a paper in Nature, Tou et al. replace dsDNA molecules with ones of circular single-stranded DNA (cssDNA) that are recognizable by recombinases while evading immune detection, enabling the non-viral delivery of kilobase-scale cargo in vivo.
To make cssDNA a suitable substrate of recombinases, which bind only dsDNA, the authors preannealed a DNA oligonucleotide, called a partial-duplex integration polynucleotide, to the cssDNA so it contains a short dsDNA binding site for the recombinase. This oligo-annealed cssDNA (oDNA) offers the combined advantages of being recognized by the recombinase and localized to the nucleus while retaining the properties of cssDNA for targeted integration and immune evasion.
