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Selection of XID target site

Selection of XID target site. -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. Generation of XID sequence specific I-Anil variants through yeast surface display libraries.

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Selection of XID target site

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  1. Selection of XID target site -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

  2. Generation of XID sequence specific I-Anil variants through yeast surface display libraries Computational design (169) Biotin STS1 STS2 I-AniI Myc HA Aga2p s s s s Aga1p Yeast surface XID N oligo-PE Ani wt oligo-APC pCTCON2 REOAni-Myc STS1 STS2 pCTCON2 REOAni-Myc Oligo PCR and yeast transformation Error prone PCR (2 rounds) Yeast tranformation to generate library Ani Myc-FITC Surface expression selection Counter Selection

  3. EP-PCR Control Input library Output library 59 mutation/7.44 kb 1 mutation/6 kb I-Anil yeast surface expression after error prone PCR Error prone PCR mutation rate

  4. XID N oligo-APC XID N oligo-APC Ani wt oligo-PE Ani wt oligo-PE XID N oligo-APC Ani wt oligo-PE XID N oligo-APC Ani wt oligo-PE Generation of Y2 Ani XID N2 Libraries XID N oligo-APC Ani wt oligo-PE Round 5 counter selection Y2 Ani XID N2 Lib14 Round 1 counter selection Y2 Ani XID N2 Lib4 Round 3 counter selection Y2 Ani XID N2 Lib10 XID N oligo-APC Ani wt oligo-PE Round 7 counter selection Y2 Ani XID N2 Lib18 Round 8 counter selection Y2 Ani XID N2 Lib21 Round 9 counter selection Y2 Ani XID N2 Lib24

  5. Titration of WT Ani, XID Lib7, 11, 18 and 23 towards XID -5 oligo and WT oligo. XID Lib23

  6. Sequence analysis from Ani XID N2 Lib24 (32 colonies)

  7. WT oligo-APC XID N Oligo-PE Biotin XID oligo I-Anil DT40 B lymphocytes Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation Selected for continuous culture and mutation I-Anil DT 40 surface display and somatic hypermutation XID oligo binding affinity

  8. Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation Oligo-PE Oligo-PE -3 oligo 5th round sorting -3 oligo 2nd round sorting WT oligo-APC Oligo-PE XID N Oligo-PE -5 oligo 4th round Sorting -5 oligo 3rd round counter selection.

  9. Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation WT oligo-APC WT oligo-APC XID N Oligo-PE XID N Oligo-PE -5 oligo 5th round counter selection. -5 oligo 4th round counter selection. WT oligo-APC WT Oligo-PE XID Oligo-PE XID Oligo-PE XID oligo 1st round counter selection.

  10. I-Anil coding sequence in DT40 cells WT WT

  11. XID oligo 6thround counter selection. 9th rounds binding sorting WT oligo-APC Side scatter XID Oligo-PE XID Oligo-PE

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