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Selection of XID(Btk) site specific I-AniI variant for gene repair in hematopoietic stem cells

Selection of XID(Btk) site specific I-AniI variant for gene repair in hematopoietic stem cells. Yupeng Wang. Rawlings Lab Center for Immunity and Immunotherapy Seattle Children's Research Institute. Selection of XID target site. -10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9.

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Selection of XID(Btk) site specific I-AniI variant for gene repair in hematopoietic stem cells

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  1. Selection of XID(Btk) site specific I-AniI variant for gene repair in hematopoietic stem cells Yupeng Wang Rawlings LabCenter for Immunity and ImmunotherapySeattle Children's Research Institute

  2. Selection of XID target site -10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9 WT-Ani: 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

  3. 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

  4. 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

  5. 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

  6. Titration of Lib23 towards XID -5 oligo and WT oligo.

  7. 18 20 22 24252627 29 31 33 35 55 57 59 61 6364 66 68 70 72 75 LVGLYEGDG Y F S I T K K G K Y L T Y E L G I E LSIKDVQLIYKIKKILGIG I V S F R K R N E I E M V A L R I R DK N HIKSK S Q T N G K Y Q S R E V R Y R KE R Q V R D Sequence analysis from Ani XID N2 Lib24 (32 colonies)

  8. Y2 Ani XID N library Flow cleavage assay Ca2+ Mg2+ WT Oligo Oligo APC XID N oligo-PE -5 XID Oligo Myc-FITC Oligo PE

  9. 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

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

  11. LVGLYEGDG Y F S I T K K G K Y L T Y E L G I E LSIKDVQLIYKIKKILGIG I V S F R K R N E I E M V A L R I R DK N HIKSK Sequence analysis from DT40 Ani XID Lib2 (-5 oligo) 17 18 20 33 42 A N Y R E

  12. Generation of SCID sequence specific I-Anil variants through B cell somatic hypermutation XID oligo-APC SCID Oligo-PE SCID -3 oligo 4thround counter selection.

  13. Generation of DT40 Ani XID yeast Libraries Lib 2 XID N oligo-PE Myc-FITC No staining DT40 yeast library Empty Vector Ani WT DT40 Coding Sequence DT40 yeast lib1 5 0 19 DT40 yeast Lib2 4 14 6

  14. On progress and future experiments Optimize the condition for yeast library cleavage selection Cleavage selection with bacterial system Iterate DT40 selection for XID and SCID sequence

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