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)

I-AniI variants working in bacteria-based cleavage assay

Positions

Clones 4 13 46 49 55 91 92 111 209 222 Suvival rate (%)

WT Leu Phe Lys Lys Ile Phe Ser Ser Val Lys < 0.2

#2

#3

#6 (Y2)

#7

#14

#16

#20

#24

#28

LT

YR

LEA

M5

Ile

Ile

Tyr

Leu

Tyr

Tyr

Tyr

Val

Tyr

Leu

Leu

Tyr

Tyr

Gln

Gln

Gln

Gln

Glu

Glu

Val

Val

Ile

Ile

Thr

Thr

Thr

Thr

Thr

Tyr

Phe

Phe

Tyr

Ala

Ala

Arg

Arg

36 ± 3.8

75 ± 9.6

95 ± 30

85 ± 16

93 ± 4.7

3.5 ± 1.2

94 ± 18

71 ± 11

90 ± 17

68 ± 11

37 ± 13

< 0.2

92 ± 10

*

*

**

**Clones resulting from random mutagenesis and selection

Clones resulting from site-directed mutagenesis

Mutations improving the cleavage activity of I-AniI

F13YF13Y

I55VI55V

S111YS111Y

90° rotation

F91IF91IS92TS92T

In vitro cleavage asssay

40

25

15

60

190

(kDa) WT Y2 M5

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.

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

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)

A 19 bp sequence within the CFTR gene is similar to the I-AniI target sequence

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

508

Goal: Generate an I-AniI variant that specifically recognizes this site

Staining identifies two variants of CFTR target site for selection experiments

Cel

l cou

nt

(% o

f max

imum

)

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

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+6Aselection

I-AniI, -2C+8C+9T

selection

WT -2C -2C+6A -2C+8C+9T

Target site variant

I-AniI target site binding

WT Site

I-AniI, WT

WT DT40

-2C -2C+6A -2C+8C+9T

I-AniI -2C+6Aselection

I-AniI, -2C+8C+9T

selection

WT -2C -2C+6A -2C+8C+9T

Target site variant

I-AniI target site binding

I-AniI target site binding

0

10

20

30

40

50

60

70

I-SceI WT Site Full CF site

Target site variant

PE

WT I-AniI Selected I-AniI

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

G33R mutation is fixed in selected populations: present in 93/96 sequenced I-AniI clones

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

MGSSHHHHHHSSGLVPRGSDLTYAYLVGLFEGDGYFSITKKGKYLTYELRIELSIKDVQLIYKIKKILGIGIVSFRKRNEIEMVALRIRDKNHLKSFILPIFEKYPMFSNKQYDYLRFRNALLSGIISLEDLPDYTRSDEPLNSIESIINTSYFSAWLVGFIEAEGCFSVYKLNKDDDYLIASFDIAQRDGDILISAIRKYLSFTTKVYLDKTNCSKLKVTSVRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 +7 +8 +9

G33R

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)

Flow cytometry analysis of CTLMA2 targets

5’ TGAGGAGGTTTCTCTGTAA 3’5’ AGAGGACCTTCATCTGTCA 3’

I-AniI WT:I-AniI CTLMA2:

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)

I-AniI

I- CreI

I-MsoI

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)