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Yupeng

Yupeng. -10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9. Ani-wt: T G A G G A G G T T T C T C T G T A A. m-XID: A G T G C C T G T T T C T C T T G A C. Biotin. 5’-TGAGGAGGTTTCTCTGTAA. 3’-ACTCCTCCAAAGAGACATT. m-wt: C. I-AniI. Myc. Aga2p. s. s. s. s.

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Yupeng

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  1. Yupeng

  2. -10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9 Ani-wt: T G A G G A G G T T T C T C T G T A A m-XID: A G T G C C TG T T T C T CTT G A C Biotin 5’-TGAGGAGGTTTCTCTGTAA 3’-ACTCCTCCAAAGAGACATT m-wt: C I-AniI Myc Aga2p s s s s Aga1p Yeast surface Engineering I-AniI HEs for XID site HA I-Anil Yeast surface display

  3. STS 3 STS 4 Ani XID C terminal Design (Jim Havranek from Baker Lab) 2 1 Myc-Ani Myc-Ani Myc-Ani Myc-Ani 3 4 5 6 Myc-Ani Myc-Ani Myc-Ani Myc-Ani Ani XID C variants yeast surface expression.

  4. Six combo error prone PCR Vector digestion Yeast transformtion to establish Ani XID-C library (9×106) First round sorting based on Myc-Ani expression.

  5. Second round sorting based on binding affinity toward Ani +6T+7G+9C. Ani wt Library w/o sorting Ani-Myc sorting Ani XID +6T+7G+9C sorting Ani wt oligo Ani XID oligo (+6T +7G +9C) Binding affinity of Ani variants toward Ani wt and Ani +6T+7G+9C oligos.

  6. STS 3 STS 4 Copy number Fold enrichment JH Design 1 2 3 4 5 6 JH Design 1 2 3 4 5 6 D P Ani XID variants sequence analysis (45 variants) Future experiments: Characterizing binding affinity and cleavage activity of individual variant. Engineering Ani XID N variants.

  7. Jordan

  8. backbone groove mutated backbone targeted mutated groove

  9. CTD mutants of I-Ani1: Selection by YSD for higher affinity to target E148D E148D E148D C150S L156R E148D C150R K155N L156R C150R L156R L156R L156R I164V Ani WT E148D L156R L156R L156R L156R C198R L156R Q171H D173N C198R 15nM 90nM 25nM 4nM 10nM

  10. YSD Kd titration for Reo’s “Y2” mutant 10nM 50nM 90nM

  11. Melissa

  12. Current Projects • Expression of I-SceI (wt coding sequence) in S. cerevisiae • Design of REO-I-SceI (Restriction Enzyme-saturated ORF) for target of canine X-SCID site • Future projects: REO-I-CreI monomer and REO-I-MsoI monomer

  13. Design of REO-I-SceI • Optimized codon usage for S. cerevisiae expression • Eliminated splice donors and acceptors • Inserted silent restriction sites around desired motifs. I-SceI

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