1. I. Selections: I-AniI for wild-type site binding/cleavage: “WT-opt” (Ryo) I-AniI towards hCF (Audrey) I-AniI towards A.gam. CTLMA2 (Ryo) II. Binding specificity profiles: I-CreI, I-MsoI, I-AniI (WT vs. wt, WT vs. lib4, WT-opt vs. wt), I-SspI III. Crystal Structures: Single Chain I-MsoI Crystallization trials continuing with I-AniI vs. lib4 I-MsoI designs (Justin Ashworth/Greg Taylor) IV.I-AniI nickase: Metal and pH dependence, binding affinity, relative cleavage Recombination in cellulo (with Stefan Pellenz/Ray Monnat)

2. I-AniI variants working in bacteria-based cleavage assay * * ** Clones resulting from random mutagenesis and selection Clones resulting from site-directed mutagenesis

3. Mutations improving the cleavage activity of I-AniI F13Y I55V S111Y 90° rotation F91IS92T

4. In vitro cleavage asssay 40 25 15 60 190 (kDa)WTY2M5

5. CD spectrum and thermal stability of I-AniI variants Buffer : 10 mM potassium phosphate (pH 7.0) Tm : WT, 53.9 °C; Y2, 52.6 °C; M5, 43.5 °C.

6. Future Work Binding assay to AniI wt and Lib4 sites GFP recombination assay with I-AniI/Y2 (Crystallization of I-AniI/Y2 with DNA) Engineering of I-AniI variants specifically cleaving CTLMA2 site

7. I. Selections: I-AniI for wild-type site binding/cleavage: “WT-opt” (Ryo) I-AniI towards hCF (Audrey) I-AniI towards A.gam. CTLMA2 (Audrey) II. Binding specificity profiles: I-CreI, I-MsoI, I-AniI (WT vs. wt, WT vs. lib4, WT-opt vs. wt), I-SspI III. Crystal Structures: Single Chain I-MsoI Crystallization trials continuing with I-AniI vs. lib4 I-MsoI designs (Justin Ashworth/Greg Taylor) IV.I-AniI nickase: Metal and pH dependence, binding affinity, relative cleavage Recombination in cellulo (with Stefan Pellenz/Ray Monnat)

8. A 19 bp sequence within the CFTR gene is similar to the I-AniI target sequence  508  Goal: Generate an I-AniI variant that specifically recognizes this site

9. Staining identifies two variants of CFTR target site for selection experiments Cell count (% of maximum) dsOligo-BT:SAv-PE WT-2C-2C+6A-2C+8C+9T-2C+6A+8C+9T WT DT40 I-AniI (3D5) WT Site-2C-2C+6A-2C+8C+9T-2C+6A+8C+9T

10. I-AniI, WT WT DT40 I-AniI population binds selected target oligos with high affinity after 4 iterative mutation/selection rounds WT Site-2C-2C+6A-2C+8C+9T I-AniI -2C+6A selection I-AniI, -2C+8C+9T selection WT-2C-2C+6A-2C+8C+9T Target site variant I-AniI target site binding

11. WT Site I-AniI, WT WT DT40 -2C-2C+6A-2C+8C+9T I-AniI -2C+6A selection I-AniI, -2C+8C+9T selection WT-2C-2C+6A-2C+8C+9T Target site variant I-AniI target site binding Selected I-AniI population maintains ability to discriminate between similar DNA sequences I-AniI population binds selected target oligos with high affinity after 4 iterative sorting/selection rounds

12. G33R mutation is fixed in selected populations: present in 93/96 sequenced I-AniI clones MGSSHHHHHHSSGLVPRGSDLTYAYLVGLFEGDGYFSITKKGKYLTYELR IELSIKDVQLIYKIKKILGIGIVSFRKRNEIEMVALRIRDKNHLKSFILP IFEKYPMFSNKQYDYLRFRNALLSGIISLEDLPDYTRSDEPLNSIESIIN TSYFSAWLVGFIEAEGCFSVYKLNKDDDYLIASFDIAQRDGDILISAIRK YLSFTTKVYLDKTNCSKLKVTSVRSVENIIKFLQNAPVKLLGNKKLQYLL WLKQLRKISRYSEKIKIPSNY -10-9-8-7-6-5-4-3-2+1+2+3+4+5+6+7+8+9 G33R

13. I. Selections: I-AniI for wild-type site binding/cleavage: “WT-opt” (Ryo) I-AniI towards hCF (Audrey) I-AniI towards A.gam. CTLMA2 (Audrey) II. Binding specificity profiles: I-CreI, I-MsoI, I-AniI (WT vs. wt, WT vs. lib4, WT-opt vs. wt), I-SspI III. Crystal Structures: Single Chain I-MsoI Crystallization trials continuing with I-AniI vs. lib4 I-MsoI designs (Justin Ashworth/Greg Taylor) IV.I-AniI nickase: Metal and pH dependence, binding affinity, relative cleavage Recombination in cellulo (with Stefan Pellenz/Ray Monnat)

14. Flow cytometry analysis of CTLMA2 targets 5’ TGAGGAGGTTTCTCTGTAA 3’ 5’ AGAGGACCTTCATCTGTCA 3’ I-AniI WT: I-AniI CTLMA2:

15. I. Selections: I-AniI for wild-type site binding/cleavage: “WT-opt” (Ryo) I-AniI towards hCF (Audrey) I-AniI towards A.gam. CTLMA2 (Audrey) II. Binding specificity profiles: I-CreI, I-MsoI, I-AniI (WT vs. wt, WT vs. lib4, WT-opt vs. wt), I-SspI III. Crystal Structures: Single Chain I-MsoI Crystallization trials continuing with I-AniI vs. lib4 I-MsoI designs (Justin Ashworth/Greg Taylor) IV.I-AniI nickase: Metal and pH dependence, binding affinity, relative cleavage Recombination in cellulo (with Stefan Pellenz/Ray Monnat)

16. I-AniI I- CreI I-MsoI

17. I. Selections: I-AniI for wild-type site binding/cleavage: “WT-opt” (Ryo) I-AniI towards hCF (Audrey) I-AniI towards A.gam. CTLMA2 (Audrey) II. Binding specificity profiles: I-CreI, I-MsoI, I-AniI (WT vs. wt, WT vs. lib4, WT-opt vs. wt), I-SspI III. Crystal Structures: Single Chain I-MsoI Crystallization trials continuing with I-AniI vs. lib4 I-MsoI designs (Justin Ashworth/Greg Taylor) IV.I-AniI nickase: Metal and pH dependence, binding affinity, relative cleavage Recombination in cellulo (with Stefan Pellenz/Ray Monnat)