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 LabCenter for Immunity and Immunotherapy
Seattle Children's Research Institute
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 Am-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
Ani wt oligo-APC
XID
N o
lig
o-P
E
3216854 bp
pCTCON2 REOAni-Myc
STS1 STS2
Oligo PCR and yeast transformation Error prone PCR (2 rounds) Yeast tranformation to generate library
3216854 bp
pCTCON2 REOAni-Myc
STS1 STS2
Computational design (169)
Yeast surface
Aga1pss
Aga2p
I-AniI
MycHA
Biotin
ss
Surface expression selection Counter Selection
Ani Myc-FITC
Generation of XID sequence specific I-Anil variants through yeast surface display libraries
Error prone PCR mutation rate
0
1
2
3
4
5
6
7
8
9
1 2
Mu
tati
on
s/kb
EP-PCR Control59 mutation/7.44 kb 1 mutation/6 kb
0
10
20
30
40
50
60
1 2
An
i Su
rfa
ce
ex
pre
ss
ion
%Input library Output library
I-Anil yeast surface expression after error prone PCR
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 1 counter selection Y2 Ani XID N2 Lib4
Generation of Y2 Ani XID N2 Libraries
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 3 counter selection Y2 Ani XID N2 Lib10
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 5 counter selection Y2 Ani XID N2 Lib14
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 7 counter selection Y2 Ani XID N2 Lib18
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 8 counter selection Y2 Ani XID N2 Lib21
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 9 counter selection Y2 Ani XID N2 Lib24
Titration of Lib23 towards XID -5 oligo and WT oligo.
18
16
14
12
10
8
6
4
2
0
MF
I
1 10 100 1000Substrate (nM)
Ani WT oligo XID -5 oligo
Sequence analysis from Ani XID N2 Lib24 (32 colonies)
Desgin site WT STS2 region WT
STS1 region 55 I A 1 Q 7 V 24
18 Y Y 3 Q 1 S 28 57 S R 25 Y 7
20 S S 3 H 1 K 1 Q 26 R 1 59 R R 7 V 1 Y 24
22 T T 32 61 R R 32
24 K N 32 63 E K 32
25 G G 32 64 I E 28 K 4
26 K K 32 66 M R 32
27 Y Y 32 68 A Q 32
29 T H 1 Q 31 70 R A 1 V 31
31 E S 31 T 1 72 R R 32
33 G R 32 Random mutation
35 E E 32 75 N N 3 D 29
122 D D 17 V 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 HIKSK18 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
Y2 Ani XID N library Flow cleavage assay
WT Oligo
Oligo PE
Oli
go
AP
C
-5 XID Oligo
Ca2+ Mg2+
Myc-FITC
XID
N o
lig
o-P
E
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
oli
go
-AP
C
Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation
XID N Oligo-PE
WT
oli
go
-AP
C
-5 oligo 5th round counter selection.
XID Oligo-PE WT Oligo-PE
XID Oligo-PE
WT
oli
go
-AP
C
XID oligo counter selection.
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 site WT
17 G G 3 A 22
18 Y Y 3 N 22
20 S S 4 Y 21
42 Q Q 3 E 22
80 K F 25
113 E E 3 K 22
121 S S 15 ? N 10 ?
232 K L 25
Sequence analysis from DT40 Ani XID Lib2 (-5 oligo)
Generation of SCID sequence specific I-Anil variants through B cell somatic hypermutation
SCID Oligo-PE
XID
oli
go
-AP
C
SCID -3 oligo 4th round counter selection.
Generation of DT40 Ani XID yeast Libraries
Myc-FITC
XID
N o
lig
o-P
E
No staining DT40 yeast library
Empty Vector Ani WT DT40 Coding SequenceDT40 yeast lib1 5 0 19 DT40 yeast Lib2 4 14 6
Lib 2
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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