1. Expanding the Pool Characterizing LAGLIDADG Homing Endonuclease Orthologs

2. Picking the ORF Regions to Use Intron ORF OK active sites 50-60+ % homology Regions homologous to Ani Optimization – Yeast expression, restriction – Surface expression?

3. Expression & Relatedness

4. Expression problems Mutagenesis & directed evolution to regain & improve expression Glycosylation changes? – Tas Tin and Vin are predicted to be lacking glycosylation in regions glycosylated in Pno, Ach, Hje, and Ani – Tas and Tin are predicted to be glycosylated at DNA binding regions Poor surface expressers but otherwise OK

5. Target Determination Target for Ani at intron/exon junctions By Homology… Central 4 are hard to predict TGAGGAGGTT T CT CTGTAAA TGGGGAGG TTT TTCAGTATC

6. Binding Vs Ani Target & Predicted Targets Conclusion: Tas, Tin, and Vin are not producing a viable surfac-expressed HE

7. Cleavage Ach Hje Pno Ani E148D Ach Hje Pno Ach Hje Pno Ani 37°C, against Predicted Targ37°C, against Ani Targ 30°C, against Predicted Targ Target Only

8. What Changes Are Responsible? Variance at DNA-interacting domains – Insertions relative to Ani

9. Differences at DNA-Binding Regions 5Å interactions as spheres Unchanged residues in green Conserved Residues in Purple +5, +9 & +10 positions (white)+2 & +5 positions (white)

10. Insertions Directly change DNA-interacting regions (left) May realign directly interacting regions (right) – Harder to obtain by directed evolution alone +5, +9 & +10 positions (white)-8 position (white)

11. Rosetta Predictions -8 A -> G K24N & T29K D73N Conserved residue changed

12. Future Directions Mutagenesis & directed evolution to improve surface expression and/or activity Comparing Ani ability to cleave predicted targets to the corresponding enzyme’s cleavage to evaluate actual changes Determine specificity with 1-off panels DNA shuffling to generate hybrid HE’s Begin to look at which changes are tolerated and which aren’t Take-home Message We can use homology searches to find functional homing endonucleases with different targets This can help us determine what AA changes affect what target specificities

13. Un-cleaved Targets No background where the cleaved band would show when targets only are run Un-cleaved Targets Cleaved Targets Ach Hje Pno Tas/Tin Vin Onu Ani E148D

14. Predicteced Glycosylated sites circled