Homeodomains (HDs) are a large family of DNA-binding domains contained in transcription factors that are most notable for regulating body development and patterning in metazoans. HDs consist of three alpha helices preceded by an N- terminal arm, where the third helix (the recognition helix) and the N-terminal arm are responsible for defining DNA-binding specificity. Here we attempted to engineer the HDs by fully randomizing positions in the recognition helix to specify each of the 64 possible 3’ triplet sites (i.e. TAANNN). We recovered HD variants that preferentially recognize or are compatible with 44 of the possible sites, a dramatic increase from the previously observed range of specificities. Many of these HD variants contain combinations of novel specificity determinants that are uncommon or absent in extant HDs, where these determinants can be grafted into alternate HD backbones with an accompanying alteration in their specificity. The identified determinates expand our understanding of HD recognition, allowing for the creation of more explicit recognition models for this family. Additionally, we demonstrate that HDs can recognize a broader range of DNA sequences than anticipated, thus raising questions about the fitness barrier that restricts the evolution HD-DNA recognition in nature. Finally, these new HD variants have utility as DNA-binding domains to direct targeting of customizable sequence-specific nuclease as demonstrated by site-specific lesions created in zebrafish. Thus HDs can guide sequence-specific enzymatic function precisely and predictably within a complex genome when used in engineered artificial enzymes.

The recognition potential of most families of DNA-binding domains (DBDs) remains relatively unexplored. Homeodomains (HDs), like many other families of DBDs, display limited diversity in their preferred recognition sequences. To explore the recognition potential of HDs, we utilized a bacterial selection system to isolate HD variants from a randomized library that are compatible with each of the 64 possible 3' triplet sites (i.e. TAANNN). The majority of these selections yielded sets of HDs with overrepresented residues at specific recognition positions, implying the selection of specific binders. The DNA-binding specificity of 151 representative HD variants was subsequently characterized, identifying HDs that preferentially recognize 44 of these target sites. Many of these variants contain novel combinations of specificity determinants that are uncommon or absent in extant HDs. These novel determinants, when grafted into different HD backbones, produce a corresponding alteration in specificity. This information was used to create more explicit HD recognition models, which can inform the prediction of transcriptional regulatory networks for extant HDs or the engineering of HDs with novel DNA-recognition potential. The diversity of recovered HD recognition sequences raises important questions about the fitness barrier that restricts the evolution of alternate recognition modalities in natural systems.