1. Monomerization of Homing Endonucleases
Targets: CreI, MsoI, and CeuI.
Goal: Generation of catalytically active monomeric HEs by connecting two
copies of protein with a linker.

2. Monomerization of Homing Endonucleases
Gene synthesis
Protein Science (2003), 12:
Library size: ~1×106
J.A.C.S. (2006), 128:

3. Monomeric Cre3 (mCre) and Mso24 (mMso)
Protein
Linker Sequence
mCreI
TGSGSGSKSQAVAHPTDGQRDFGAKGSGSGSGT
mMsoI
TGSGSGSKHPTLTLPTTTSQENLPNGSGSGSGT
With a 33 Aa linker between two HE domains, both mCre and mMso show comparable site-specific cleavage activity as WT HEs.
Size-exclusion chromatography confirms the monomeric state of both proteins .

4. Biochemical Characterization by Circular Dichroism (CD)
CreWT and mCre show distinctive secondary structures and thermostability profile by far-UV CD.
Protein
Tm (ºC)
CreWT
66.81±0.01
mCre
56.46±0.07

5. Biochemical Characterization by Circular Dichroism (CD)
MsoWT and mMso show similar secondary structures and thermostability profile.
Protein
Tm (ºC)
MsoWT
54.30±0.01
mMso
56.23±0.08

6. Thermodynamic Values of mCre by ITC
CreWT
mCre
Protein KD nM ∆H
kcal/mol ∆S
cal/mol/deg
-T∆S ∆G
CreWT
13.3±2.6
19.6±0.20
101
-30.6
-11
mCre
25.8±4.7
26.7±0.59
123±1.0
-37.3
-11.5

7. Thermodynamic Values of mMso by ITC
MsoWT1
mMso
Protein KD nM ∆H
kcal/mol ∆S
cal/mol/deg
-T∆S ∆G
MsoWT1
21±5.0
12.7±0.27
77±1.1
-23.3
-10.6
Mso
40.2±9.6
14.7±0.4
82.4
-25.0
-10.3
1. Eastberg et. al. Nucleic Acid Res. (2007), 35(21):

8. Competitive Binding Matrix
KaA
KaWT
(FNC-FA)×FWT
FA×(FNC-FWT) =
Ka = association constant
F = fluorescence
A = oligos with one bp mismatch
WT = WT oligos
NC = random oligos
Lei Zhao

9. Competitive Binding Matrix of MsoWT and mMso
Lindsey Doyle
mMso
MsoWT and mMso show similar binding specifities.

10. Site Specificity by Ultra-deep Sequencing
probabilistic site models/ better PSSMs
Solexa sequencing
Argast et. al. J Mol Biol. (1998), 280:

11. Sequential Enrichment of Cleavage-Sensitive Target Sites
In each round, 1 pmol plasmid DNA was digested at 37ºC for 1 hour in presence of 20 pmol enzyme.
After five rounds of enrichment, the majority of library are cleavage-sensitive.

12. Summary
Two monomeric I-CreI (mCre) and I-MsoI (mMso) were selected, and show comparable in vitro cleavage activity as WT enzymes.
mCre shows distinctive structural features and DNA binding.
mMso shows similar structural, biochemical characteristics as I-MsoI.
Future Works
Crystallization of mCre and mMso.
Incorporate Mso mutations in silico designed by Justin into mMso, and validate the cleavage of asymmetric DNA target site by mMso mutants.
High-throughput sequencing and analysis of cleavage sensitive libraries by CreWT, MsoWT, and their monomeric version, and generate PSSM (position specific search matrix) for each individual enzyme.
Search for potential target sites in genes of interest using new PSSMs.
Engineering of Cre towards physiologically relevant target sites, and incorporate into monomeric version.