1. Stoddard Lab Immediate Goals (Nov-Dec 2007) Optimization of I-AniI wild-type site recognition/I-AniI stability and generation of in vivo activity (bacterial and eukaryotic) Ryo Takeuchi and Audrey McConnell-Smith Selection experiments towards Anopheles CTLMA2 and hCFTR gene targets: Ryo Takeuchi and Audrey McConnell-Smith High-throughput “bindability” assay Lei Zhao Development of homing endonuclease ‘Nickase’: recombinase? Audrey Improved I-AniI / DNA crystallization conditions (All) I-AniI apo enzyme structure at high resolution Audrey McConnell-Smith

2. Selection of I-AniI mutant working in bacteria Transform pEndo plasmid into the competent cells harboring pCcdB plasmid. Grow the transformants with carbenicillin (Cb) and L-arabinose (Ara) for 4 h. Spread them on the plates containing Cb, Ara and IPTG. E. coli HE site lacZ-ccdB pCcdB chl r pEndo cb r HE gene pBAD Cb/Ara/IPTG plates Cb + plac HE site AraIPTG Ara

3. I-AniI containing 3 mutations (F13L/K46Q/S92T) improves cleavage of WT site in bacteria HE gene on pEndo plasmid I-AniI wt I-AniI/LQT HE sites on pCcdB : AniI wt sites AniI -9A/-8G sites Ara/IPTG - + ~ 80-90 % of the transformants harboring I-AniI/LQT gene could survive on the Ara/IPTG plates.

4. Position of the mutations F13L K46Q S92T Rotate 90° F13L K46Q S92T

5. Future work 1.Continue screening for additional of I-AniI mutants optimized for cleavage of wild type site 2.Compare activity with Lib4 (hypercleavable) site 3.Characterize the mutations essential for the robust activity of I-AniI: stabilizing? better expression? tighter binding? faster cleavage? 4.Selection of I-AniI cleaving the CTLMA2 -4C substrate 5’-TGAGGAGGTTTCTCTGTAAA-3’ 5’-AGAGGACCTTCATCTGTCAA-3’ AniI wt : CTLMA2 : -10 +1 +10

6. Future work 5’-TGAGGAGGTTTCTCTGTAAA-3’ 5’-AGAGGACCTTCATCTGTCAA-3’ AniI wt : CTLMA2 : -10 +1 +10

7. CFTR gene targeting  508

9. Asp 16 Glu 148 Leu 36 Lys 227 Asp 40 (Q171) (K94) (G174) Uncut Linear Nicked Linear Supercoiled HmuI WT Q171K K227M Q171K +

10. High-throughput fluorescent competitive binding assay for determining specificity of homing endonucleases