monnat lab progress monomerizations of cre, mso (hui) 2 nd round designs vs. anopheles targets...

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.