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I. Selections: I-AniI for wild-type site binding/cleavage: “WT-opt” (Ryo)

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:

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I. Selections: I-AniI for wild-type site binding/cleavage: “WT-opt” (Ryo)

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  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) 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 F91I S92T 90° rotation F13Y I55V S111Y

  4. In vitro cleavage asssay (kDa) WT Y2 M5 190 60 40 25 15

  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) 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 WT -2C -2C+6A -2C+8C+9T -2C+6A+8C+9T WT DT40 Cell count (% of maximum) I-AniI (3D5) dsOligo-BT:SAv-PE WT Site -2C -2C+6A -2C+8C+9T -2C+6A+8C+9T

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

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

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

  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) 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 I-AniI WT: I-AniI CTLMA2: 5’ TGAGGAGGTTTCTCTGTAA 3’ 5’ AGAGGACCTTCATCTGTCA 3’

  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) 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) I-AniI nickase: Metal and pH dependence, binding affinity, relative cleavage Recombination in cellulo (with Stefan Pellenz/Ray Monnat)

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