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

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    03-Feb-2016
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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: - PowerPoint PPT Presentation

Transcript of 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-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