monnat lab progress monomerizations of cre, mso (hui) 2 nd round designs vs. anopheles targets...
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![Page 1: Monnat Lab progress monomerizations of Cre, Mso (Hui) 2 nd round designs vs. Anopheles targets (Umut) in vivo recombination reporter assays (Ray, Hui)](https://reader035.vdocument.in/reader035/viewer/2022062301/5697bf9f1a28abf838c94ef8/html5/thumbnails/1.jpg)
Monnat Lab progress
monomerizations of Cre, Mso (Hui)
2nd round designs vs. Anopheles targets
(Umut)
in vivo recombination reporter assays
(Ray, Hui)
completed/sequence-verified reporters
completed expression vectors
functional analyses of Sce/Ani/Cre
to discuss: expression vectors/target
cells
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Monomerization of Homing Endonucleases
Gene synthesis
Scalley-Kim M. et. al. Protein Science (2003), 12: 197-206
Doyon JB et. al. J.A.C.S. (2006), 128: 2477-84.
Library size: ~1×106
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The Length and Aa Distribution of Linkers
Aa distribution in Cre Library
0
2
4
6
8
10
12
Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr
Amino AcidsPe
rcen
tage
(%)
Naïve
Selected
Aa distribution in Mso library
0
5
10
15
20
25
Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr
Amino acids
Perc
enta
ge(%
)
Naïve
Selected
• 19 various linkers were identified from Cre library with length from 13 Aa to 73 Aa.
• 13 various linkers were identified from Mso library with length at 13 and 33 Aa.
![Page 4: Monnat Lab progress monomerizations of Cre, Mso (Hui) 2 nd round designs vs. Anopheles targets (Umut) in vivo recombination reporter assays (Ray, Hui)](https://reader035.vdocument.in/reader035/viewer/2022062301/5697bf9f1a28abf838c94ef8/html5/thumbnails/4.jpg)
In Progress
• Structural analysis by crystallography.
• Biochemical study:
Binding by isothermal titration calorimetry (ITC) and fluorescence anisotropy.
Thermal stability by circular dichroism (CD).
Cleavage specificity matrix
• In vivo function assay using pDR-GFP recombination system.
•Verification of monomeric HEs as novel platform for future engineering.
Introduction of mutations with known specificity changes in single domain.
Construction of Cre and Mso heterodimer.
![Page 5: Monnat Lab progress monomerizations of Cre, Mso (Hui) 2 nd round designs vs. Anopheles targets (Umut) in vivo recombination reporter assays (Ray, Hui)](https://reader035.vdocument.in/reader035/viewer/2022062301/5697bf9f1a28abf838c94ef8/html5/thumbnails/5.jpg)
TgcgcGTcgCTCAGGgcAaTGAA GELSOLIN
CgAtcGcaaCGCAAGTGCaGGAT CHEERIO
TTctcCcagCGCAAcTACGTGAA YOLKLESS
CAAgAGccatTTAAaAATCTGAA HIV1
WT: CCAAACTGTCTCAAGTTCCGGCG
Engineering Targets
9.14 kb
43.6 kb
7.14 kb
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TgcgcGTcgCTCAGGgcAaTGAA GELSOLIN 1. CgcgcCTGTCTCAAGTTCCGGCG 2. CCAAACTcgCTCAAGTTCCGGCG 3. CCAAACTGTCTCAAGgcaaGGCG
CgAtcGcaaCGCAAGTGCaGGAT CHEERIO 4. CCAtcCTGTCTCAAGTTCCGGCG 5. CCAAACcaaCTCAAGTTCCGGCG
TTctcCcagCGCAAcTACGTGAA YOLKLESS 6. CCctcCTGTCTCAAGTTCCGGCG 7. CCAAACcagCTCAAGTTCCGGCG
CAAgAGccatTTAAaAATCTGAA HIV1 8. CCAAACccatTCAAGTTCCGGCG
WT: CCAAACTGTCTCAAGTTCCGGCG
Engineering Targets
![Page 7: Monnat Lab progress monomerizations of Cre, Mso (Hui) 2 nd round designs vs. Anopheles targets (Umut) in vivo recombination reporter assays (Ray, Hui)](https://reader035.vdocument.in/reader035/viewer/2022062301/5697bf9f1a28abf838c94ef8/html5/thumbnails/7.jpg)
Cleavage Assays
substrateproducts
controls+ –
+ –
substrateproducts
Design 1
Design 2
Design 4
Design 5
Design 7
Design 8
3 kb
3 kb
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Universal pDRGFP/HEG-specific reporter plasmids
Universal pDRGFP/HEG-specific reporter plasmids
sequence verified:
pDRGFP-ScepDRGFP-PpopDRGFP-AnipDRGFP-AniLib4pDRGFP-Dre3pDRGFP-Dre4pDRGFP-MsopDRGFP-CeupDRGFP-CpapDRGFP-DpapDRGFP-Cre (Hui)
Xba SpeIHE cut site
XhoI/KpnI/SacI
AflII
carb
puro
GFP 5'
iGFP 3'
![Page 9: Monnat Lab progress monomerizations of Cre, Mso (Hui) 2 nd round designs vs. Anopheles targets (Umut) in vivo recombination reporter assays (Ray, Hui)](https://reader035.vdocument.in/reader035/viewer/2022062301/5697bf9f1a28abf838c94ef8/html5/thumbnails/9.jpg)
HEG expression vectorsHEG expression vectors
HEG pCS2 pCMVother
I-SceI: pCSce, pCMVSce -
I-AniI: pCSAni - pHyperAniK
(pCMV+pEF)
I-PpoI: - pCNPpo6 -
I-CreI: pCSCre pCMVCre3 -
I-SpomI: pCSSpom pCMVSpom -
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293T – 72h after transfection293T – 72h after transfection
-/pEGFP-C1 (FuGENE6)
GFP
PI (dead)
-/pEGFP-C1/CaPO4
15.5% 56.9%
-/pDRGFPuniv
0.5%
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pCMVSce/pDRGFPSce pCSAni/pDRGFPAni pCSAni/pDRGFPLib4
293T – 72h after transfection (FuGene6)293T – 72h after transfection (FuGene6)
1.5% 0.2% 0.4%
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%GFP (hrs)
cells/plasmid(s) tf 48 72 96hrs
293T CaPO4 0.7 0.0
+ EGFP-C1 79.0
15.5 19.8
66.3
+ DRGFPuni 0.0 0.7
+ Sce/DRSce 1.3 11.7
+ Ani/DRAni 0.1 1.7
+ EGFP-C1 FuGENE6 56.9 51.8
+ DRGFPuni 0.5 0.8
+ Sce/DRSce 1.5 3.2
+ Ani/DRAni 0.2 0.5
+ Ani/DRLib4Ani 0.4 0.7
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In vivo Cre Cleavage-Mediated Recombination
-/- pEGFP pDRGFP-Cre
pDRGFP-Cre/pCSCreWT pDRGFP-Cre/pCSCreD20N
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Splice site prediction in HE ORFsSplice site prediction in HE ORFs
Prediction of cryptic splice sites in HE ORFs using• Alternative splice site predictor (http://
www.es.embnet.org/~mwang/assp.html)• Berkeley Drosophila genome project splice site prediction (http://
www.fruitfly.org/seq_tools/splice.html)• NetGene2 (http://www.cbs.dtu.dk/services/NetGene2/)• GenScan (http://genes.mit.edu/GENSCAN.html)• TIGR Gene splicer (http://www.tigr.org/tdb/GeneSplicer/gene_spl.html)
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Splice site prediction for I-AniI (JO1387)Splice site prediction for I-AniI (JO1387)
AFN conf.AFN conf. AFAFAFAF
314 471
AFNAFN
const.const. crypt.crypt.sdsd
sasa
31
AFAF
337
AFAF
424
AFNAFN
AFAF AFAF AFAF
543 614 67272
764764
A(SSP)A(SSP)F(ruitfly)F(ruitfly)N(etGene2)N(etGene2)const.const. crypt.crypt.