Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

Despite recent progress in the ability to manipulate the genomes of eukaryotic cells[1][1]–[3][2], there is still no effective and practical method to precisely integrate large synthetic DNA constructs into desired chromosomal sites using a programmable integrase. Serine integrases can perform the necessary molecular steps[4][3], but only if their natural target site is first installed into the recipient genome by other methods. A more elegant approach would be to directly reprogram the serine integrase itself to target a desired site endogenous to the genome that is different from the natural recognition site of the integrase[5][4]. Here, we describe the development of a platform of Modular Integrases (the MINT platform), a versatile protein-guided genome editing method that can facilitate site-directed targeted integration of synthetic DNA into chromosomal sites. Through a combination of structural modeling, directed evolution, and screening in human cells we have reprogrammed the specificity of the serine integrase Bxb1. We then utilized these reprogrammed Bxb1 variants to enable precise integration of kilobase-sized constructs into multiple endogenous locations within the human genome with up to 35% efficiency and promising genome-wide specificity. We demonstrate the therapeutic potential of the MINT platform by retargeting Bxb1 to the human TRAC and AAVS1 loci where wild-type Bxb1 has no detectable activity. ### Competing Interest Statement All authors contributed to this work as full-time employees of Sangamo Therapeutics. Sangamo Therapeutics has filed patent applications regarding Integrase systems described in this study, listing F.F., S.A.-F., N.A.S., and J.C.M. as inventors. [1]: #ref-1 [2]: #ref-3 [3]: #ref-4 [4]: #ref-5