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

Yupeng Wang

Rawlings LabCenter for Immunity and ImmunotherapySeattle Children's Research Institute


Selection of XID target site repair in hematopoietic stem cells

-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


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


EP-PCR Control yeast surface display libraries

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


XID N oligo-APC yeast surface display libraries

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



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)


Y2 Ani XID N library Flow cleavage assay 55 57 59 61 6364 66 68 70 72 75

Ca2+ Mg2+

WT Oligo

Oligo APC

XID N oligo-PE

-5 XID Oligo

Myc-FITC

Oligo PE


WT oligo-APC 55 57 59 61 6364 66 68 70 72 75

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


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.


LVGLYEGDG B cell somatic hypermutationY 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


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.


Generation of DT40 Ani XID yeast Libraries B cell somatic hypermutation

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


On progress and future experiments B cell somatic hypermutation

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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