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Expanding the Pool

This study focuses on the optimization of LAGLIDADG homing endonuclease orthologs by investigating open reading frame (ORF) regions, active sites, and target specificity. We explore homology relationships, yeast expression, and challenges related to surface expression and intron/ORF expression problems. Our findings involve mutagenesis and directed evolution strategies to enhance expression and activity. We predict glycosylation impacts on DNA-binding regions and analyze structural differences affecting target specificity, ultimately aiming to identify tolerable changes.

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Expanding the Pool

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  1. Expanding the Pool Characterizing LAGLIDADG Homing EndonucleaseOrthologs

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

  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 Targ 37°C, against AniTarg 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. Take-home Message Future Directions • We can use homology searches to find functional homing endonucleaseswith different targets • This can help us determine what AA changes affect what target specificities • 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

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