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-SspIIII. Crystal Structures:Single Chain I-MsoICrystallization trials continuing with I-AniI vs. lib4I-MsoI designs (Justin Ashworth/Greg Taylor)I-AniI nickase:Metal and pH dependence, binding affinity, relative cleavageRecombination in cellulo (with Stefan Pellenz/Ray Monnat)

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

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

Mutations improving the cleavage activity of I-AniIF13YI55VS111Y90 rotationF91IS92T

In vitro cleavage asssay40251560190(kDa)WTY2M5

CD spectrum and thermal stability of I-AniI variantsBuffer : 10 mM potassium phosphate (pH 7.0)

Tm : WT, 53.9 C; Y2, 52.6 C; M5, 43.5 C.

Future WorkBinding assay to AniI wt and Lib4 sitesGFP 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-SspIIII. Crystal Structures:Single Chain I-MsoICrystallization trials continuing with I-AniI vs. lib4I-MsoI designs (Justin Ashworth/Greg Taylor)I-AniI nickase:Metal and pH dependence, binding affinity, relative cleavageRecombination in cellulo (with Stefan Pellenz/Ray Monnat)

A 19 bp sequence within the CFTR gene is similar to the I-AniI target sequence508Goal: Generate an I-AniI variant that specifically recognizes this site

Staining identifies two variants of CFTR target site for selection experimentsCell count (% of maximum)dsOligo-BT:SAv-PEWT-2C-2C+6A-2C+8C+9T-2C+6A+8C+9TWT DT40I-AniI (3D5)WT Site-2C-2C+6A-2C+8C+9T-2C+6A+8C+9T

I-AniI population binds selected target oligos with high affinity after 4 iterative mutation/selection roundsI-AniI, WTWT DT40WT Site-2C-2C+6A-2C+8C+9TI-AniI -2C+6AselectionI-AniI, -2C+8C+9T selectionWT-2C-2C+6A-2C+8C+9TTarget site variantI-AniI target site binding

WT SiteI-AniI, WTWT DT40-2C-2C+6A-2C+8C+9TI-AniI -2C+6AselectionI-AniI, -2C+8C+9T selectionWT-2C-2C+6A-2C+8C+9TTarget site variantI-AniI target site bindingSelected I-AniI population maintains ability to discriminate between similar DNA sequencesI-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 clonesMGSSHHHHHHSSGLVPRGSDLTYAYLVGLFEGDGYFSITKKGKYLTYELRIELSIKDVQLIYKIKKILGIGIVSFRKRNEIEMVALRIRDKNHLKSFILPIFEKYPMFSNKQYDYLRFRNALLSGIISLEDLPDYTRSDEPLNSIESIINTSYFSAWLVGFIEAEGCFSVYKLNKDDDYLIASFDIAQRDGDILISAIRKYLSFTTKVYLDKTNCSKLKVTSVRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY

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-SspIIII. Crystal Structures:Single Chain I-MsoICrystallization trials continuing with I-AniI vs. lib4I-MsoI designs (Justin Ashworth/Greg Taylor)I-AniI nickase:Metal and pH dependence, binding affinity, relative cleavageRecombination in cellulo (with Stefan Pellenz/Ray Monnat)

Flow cytometry analysis of CTLMA2 targets5 TGAGGAGGTTTCTCTGTAA 35 AGAGGACCTTCATCTGTCA 3I-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-SspIIII. Crystal Structures:Single Chain I-MsoICrystallization trials continuing with I-AniI vs. lib4I-MsoI designs (Justin Ashworth/Greg Taylor)I-AniI nickase:Metal and pH dependence, binding affinity, relative cleavageRecombination 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-SspIIII. Crystal Structures:Single Chain I-MsoICrystallization trials continuing with I-AniI vs. lib4I-MsoI designs (Justin Ashworth/Greg Taylor)I-AniI nickase:Metal and pH dependence, binding affinity, relative cleavageRecombination in cellulo (with Stefan Pellenz/Ray Monnat)

Weighted position-specific scoring matrices (PSSMs) contain known information about engineerability and sequence preference at each target site position for eight homing endos.PSSM search performed on a 200 bp sequence flanking CFTR delta508 deletion. Best match is here. Differs at 7 positions.Goal: generate I-AniI variant that specifically recognizes this target siteCFTR sequence of the region surrounding the 508 mutation (depicted as ***). A 19 base pair sequence was identified (bold) that closely resembles the cognate I-AniI target sequence, below. Red, lower-case bases indicate positions that vary between the two sites. Numbers have been arbitrarily assigned to indicate base pair position within the target site.

B10 has high Ani expressionB3 has low Ani expression, similar to hypermutating cells3D5 has Ani integrated in hypermutating DT40 lambda light chain locus.-2C ~ 1/2 WT-2C+6A~1/4 WT-2C+8C+9T