Expanding the DNA editing toolbox: novel lambda integrase variants targeting microalgal and human genome sequences

Abstract

AbstractRecombinase enzymes are extremely efficient at integrating very large DNA fragments into target genomes. However, intrinsic sequence specificities curtail their use to DNA sequences with sufficient homology to endogenous target motifs. Extensive engineering is therefore required to broaden applicability and robustness. Here, we describe the directed evolution of novel lambda integrase variants capable of editing exogenous target sequences identified in the diatomPhaeodactylum tricornutumand the algaeNannochloropsis oceanica. These microorganisms hold great promise as conduits for green biomanufacturing and carbon sequestration. The evolved enzyme variants show >1000-fold switch in specificity towards the non-natural target sites when assayedin vitro. A single-copy target motif in the human genome with homology to theNannochloropsis oceanicasite can also be efficiently targeted using an engineered integrase, bothin vitroand in human cells. The developed integrase variants represent useful additions to the DNA editing toolbox, with particular application for targeted genomic insertion of large DNA cargos.