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. Procedures Gene Synthesis Two copies of ORFs (~65% identity) optimized based on E.coli codon usage with an internal MCS was synthesized ACCGGT ACTAGTG GGTACC AgeI SpeI KpnI Insertion of a linker library with 60 to 80 amino acid residues between two copies of ORFs. High-throughput screening or/and in vivo selection to identify active HEs in monomeric form. Characterization of individual selectants.

3. The Linker Library Scalley-Kim M. et. al. Protein Science (2003), 12: 197-206 SH2 domain V 170 G 175 Random sequences encoding 60-120 amino acids After three-round selection against SH2 ligand using phage display, a group of peptides with 60-80 amino acid residues were identified to have minimal effects on SH2 domain stability.

4. In vivo Selection Doyon JB et. al. J.A.C.S. (2006), 128: 2477-84.

5. In Vivo Selection HEs Positive Selection (Active HEs) Background (Inactive HEs) SceI a 20-40%2.0×10 -5 CreI30-40%1.5×10 -4 MsoI~30%1.0×10 -4 Control experiments a Doyon JB et. al. J.A.C.S. (2006), 128: 2477-84 Note: Two copies of target site were introduced into pCcdB vector.

6. Linkers Length of Monomeric HEs 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.

7. The Composition of Randomized Portion of the Linkers Ala, Lys, Asn, Pro, and Thr dominant.

8. The Composition of Randomized Portion of the Linkers Arg is selected for, and Gly is against.

9. The Composition of Randomized Portion of the Linkers Thr is selected for, and Gly, Ser are against.

10. In vitro Cleavage 14 monomeric Cres, and 8 monomeric Msos were cloned into expression vectors, respectively. All but one (Msomono#96) show in vitro cleavage activity

11. Verification of Protein Molecular Organization Inactive Active

12. In vitro Cleavage of Single Active Site Knock-out Monomeric HEs Single active site knock-out mutation eliminate the cleavage activity in Cremono#15, Msomono#24 and #27 completely, while partially in Cremono#6. All single active site knock-out mutants show nicking activity against supercoiled substrate.

13. In vitro Cleavage of Single Active Site Knock-out Monomeric HEs Monomeric HEs with 13 Aa linker show non-specific cleavage. Monomeric Cre with 53 Aa linker shows cleavage activity, while the one with 73 Aa linker doesn’t.

14. Conclusion Majority of monomeric HEs show in vitro cleavage activity, validating the in vivo selection system. Monomeric Hes with different linkers behave differently in term of the oligomeric organization. Future Works: Complete the cleavage profile of single active site knock-out mutants. In vivo activity in human cells using DR-GFP reporter system. Structural study.

15. In vitro Cleavage WT CreI and MsoI were expressed from pET16b vector in BL21(DE3). Monomeric CreI and MsoI were expressed from pBAD vector in TOP10. Proteins were purified Ni-NTA kit (Qiagen).

16. Monomeric CreI linkers LinkerSequenceFrequency #2 TGSGSGSTNMKPPVRAFEPTGVRSRGSGSGSGT (33Aa)6/84 #3 TGSGSGSKSQAVAHPTDGQRDFGAKGSGSGSGT (33Aa)5/84 #6 TGSGSGSKPAGGDAPRLMQGVNRIDGSGSGSGT (33Aa)19/84 #7 TGSGSGSGSGSGT (13Aa)29/84 #14 TGSGSGSNPRNSPNSKTSMPIDVNNGSAYSMQSNRGYVKEEYLHRGSGS GSGT (53Aa) 1/84 #15 TGSGSGSKTKNMSPKANIERTPENKGSGSGSGT (33Aa)7/84 #19 TGSGSGSSTKERTNLKDNMTIDKPRGSGSGSGT (33Aa)1/84 #45 TGSGSGSKDVTQANRTYIPRENASRGSGSGSGT (33Aa)1/84 #48 TGSGSGSTDQAGHDPGAKTAKPMLGGSGSGSGT (33Aa)1/84 #53 TGSGSGSNYAAKPIPSAGQLETSHNGSGSGSGT (33Aa)3/84 #56 TGSGSGSIPQTQFHLVLGAAATRDNGSGISETNPRDPTQVSDKNIGSTVT GQVVRTDSLEENKANGSGSGSGT (73Aa) 2/84 #65 TGSGSGSKTKNMSPSANIERTPDNKGSGSGSGT (33Aa)1/84 Continued

17. LinkerSequenceFrequency #81 TGSGSGSKYEGKAILSAGQLDTSYKGSGSGSGT (33Aa)1/84 #90 TGSGSGSNNKSSHPQGDVEQKHQHSGSGSGSGT (33Aa)1/84 #102 TGSGSGSTSARLYPQTTATMNDSTMGSGSGSGT (33Aa) 1/84 #119 TGSGSGSNPAMLADPKNTGLATGAIGSGSGSGT (33Aa) 1/84 #121 TGSGSGSNDTEMSSWTAERRTPRPTGSGSGSGT (33Aa) 1/84 #124 TGSGSGSNPGVRSPRNNDLPDHRLIGSGSGSGT (33Aa) 1/84 #125 TGSGSGSNAGNLPSRENNTSKHSAEGSGSGSGT (33Aa) 2/84 Monomeric CreI linkers

18. Monomeric MsoI linkers LinkerSequenceFrequency #3 TGSGSGSTAAKPPVRTTDGMESTFMGSGSGSGT(33Aa)1/57 #5 TGSGSGSGSGSGT(13Aa)15/57 #14 TGSGSGSAYTTTTDEAPTLVKPRHNGSGSGSGT (33Aa)1/57 #15 TGSGSGSKPTALNPWNIDRTTIPAKGSGSGSGT (13Aa)6/57 #24 TGSGSGSKHPTLTLPTTTSQENLPNGSGSGSGT (33Aa)3/57 #25 TGSGSGSRFAGESHVNNTTKTTKLEGSGSGSGT (33Aa)9/57 #27 TGSGSGSKTKNPHPENPGQSMTQAKGSGSGSGT (33Aa)1/57 #28 TGSGSGSRFAGESHVNNTTKTTKLEGSGSGSGT (33Aa)3/57 #29 TGSGSGSTHTTRHNRTPTAPNYRPIGSGSGSGT (33Aa)1/57 #43 TGSGSGSGFANKYNVDHNPLSNMNSGSGSGSGT (33Aa)1/57 #55 TGSGSGSKTKNPHPWNPDRSTTPAKGSGSGSGT (33Aa)1/57 #70 TGSGSGSTTQAPPTMTYTRGVATTDGSGSGSGT (33Aa)1/57 #96 TGSGSGSNLGAENAQSASQKDDALRGSGSGSGT (33Aa)1/57