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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
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
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.
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.
TgcgcGTcgCTCAGGgcAaTGAA GELSOLIN
CgAtcGcaaCGCAAGTGCaGGAT CHEERIO
TTctcCcagCGCAAcTACGTGAA YOLKLESS
CAAgAGccatTTAAaAATCTGAA HIV1
WT: CCAAACTGTCTCAAGTTCCGGCG
Engineering Targets
9.14 kb
43.6 kb
7.14 kb
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
Cleavage Assays
substrateproducts
controls+ –
+ –
substrateproducts
Design 1
Design 2
Design 4
Design 5
Design 7
Design 8
3 kb
3 kb
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'
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 -
293T – 72h after transfection293T – 72h after transfection
-/pEGFP-C1 (FuGENE6)
GFP
PI (dead)
-/pEGFP-C1/CaPO4
15.5% 56.9%
-/pDRGFPuniv
0.5%
pCMVSce/pDRGFPSce pCSAni/pDRGFPAni pCSAni/pDRGFPLib4
293T – 72h after transfection (FuGene6)293T – 72h after transfection (FuGene6)
1.5% 0.2% 0.4%
%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
In vivo Cre Cleavage-Mediated Recombination
-/- pEGFP pDRGFP-Cre
pDRGFP-Cre/pCSCreWT pDRGFP-Cre/pCSCreD20N
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)
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.