Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization

LAGLIDADG homing endonucleases (meganucleases) are sequence-specific DNA cleavage enzymes used for genome engineering. Recently, meganucleases fused to transcription activator-like effectors have been demonstrated to efficiently introduce targeted genome modifications. However, retargeting meganucleases to genomic sequences of interest remains challenging because it usually requires extensive alteration of a large number of amino acid residues that are situated in and near the DNA interface. Here we describe an effective strategy to extensively redesign such an extensive biomolecular interface. Well-characterized meganucleases are computationally screened to identify the best candidate enzyme to target a genomic region; that protein is then redesigned using iterative rounds of in vitro selections within compartmentalized aqueous droplets, which enable screening of extremely large numbers of protein variants at each step. The utility of this approach is illustrated by engineering three different meganucleases to cleave three human genomic sites (found in two exons and one flanking intron in two clinically relevant genes) and a fourth endonuclease that discriminates between single-nucleotide polymorphism variants of one of those targets. Fusion with transcription activator-like effector DNA binding domains significantly enhances targeted modification induced by meganucleases engineered in this study. Simultaneous expression of two such fusion endonucleases results in efficient excision of a defined genomic region.