Modular determinants of cleavage preference in GIY-YIG nucleases revealed by block-based DNA shuffling and directed evolution

GIY-YIG homing endonucleases are mobile genetic elements that are widespread in phage, bacterial, and organellar genomes. Their modular architecture and sequence-tolerant DNA cleavage properties likely represent evolutionary adaptations to tolerate genetic drift at target sites and enable target-site switching. To investigate modular determinants of GIY-YIG nuclease cleavage preference, we constructed 128 chimeric nucleases by shuffling structural blocks between the prototypical GIY-YIG homing endonuclease I-TevI (CNNNG motif preference) and its isoschizomer I-BmoI (NNNNG preference). Chimeras containing a swapped a-helix1 and adjacent loop exhibited altered motif preferences, highlighting this region as a modular determinant, whereas swaps in other regions disrupted activity without altering cleavage specificity. Directed evolution of non-conserved residues within this region identified a cluster (R30, K33, E36, C39) where substitutions enabled cleavage of targets not recognized by wild-type I-TevI, including variants with reprogrammed specificity toward TNNNG and GNNNG motifs. Our findings define a modular and structural basis for DNA cleavage preference in the GIY-YIG nuclease domain and suggest that recombination of structural subunits could accelerate adaptation to new target sites over evolutionary time scales. These findings further support a strategy for engineering GIY-YIG nuclease domains with expanded cleavage motif selectivity.