1. 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

2. Selection of XID target site 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 T G T T T C T C T T G A C -10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9

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

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

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

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

7. Sequence analysis from Ani XID N2 Lib24 (32 colonies) Desgin siteWT STS2 region WT STS1 region 55 I A 1Q 7V 24 18 YY 3Q 1S 28 57 S R 25Y 7 20 SS 3H 1K 1Q 26R 159 RR 7V 1Y 24 22 TT 32 61 RR 32 24 K N 32 63 E K 32 25 GG 32 64 I E 28K 4 26 KK 32 66 M R 32 27 YY 32 68 A Q 32 29 T H 1Q 31 70 R A 1V 31 31 E S 31T 1 72 RR 32 33 G R 32 Random mutation 35 EE 32 75 NN 3D 29 122 DD 17V 15 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 18 20 22 24252627 29 31 33 35 55 57 59 61 6364 66 68 70 72 75 S Q T N G K Y Q S R E V R Y R KE R Q V R D

8. Y2 Ani XID N library Flow cleavage assay WT Oligo Oligo PE Oligo APC -5 XID Oligo Ca 2+ Mg 2+ Myc-FITC XID N oligo-PE

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

10. Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation XID N Oligo-PE WT oligo-APC -5 oligo 5 th round counter selection. XID Oligo-PEWT Oligo-PE XID Oligo-PE WT oligo-APC XID oligo 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 17 18 20 33 42 A N Y R E Mutation siteWT 17 G G 3 A 22 18 YY 3 N 22 20 SS 4Y 21 42 QQ 3 E 22 80 K F 25 113 EE 3 K 22 121 SS 15 ?N 10 ? 232 K L 25 Sequence analysis from DT40 Ani XID Lib2 (-5 oligo)

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

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

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