Miller J,; McLachlan AD,; Klug A. Repetitive zinc-binding domains in the protein ... Monika A. Papworth,; Jeffrey C. Miller,; Michael P. Murphy,; Aaron Klug.
Targeted Genome Editing using Single Chain Zinc Finger Dimeric Nuclease
Muhammad Mustafa
The Graduate School Yonsei University Department of Chemistry I
Targeted Genome Editing using Single Chain Zinc Finger Dimeric Nuclease
A Master’s Thesis Submitted to the Department of Chemistry and Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Master of Chemistry
Muhammad Mustafa
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III
Contents
Contents…………………………………………………………………………….….……. (I)
List of Tables……………………………………………………………………...….…….(VI)
List of Figures……………………………………………………………………………(VIII)
Abstract …………………………………………………………………………………...(XI)
1. Introduction……………………………………………………………………1 1.1.Zinc Finger Proteins………………………………………………………….1
1.2. Genome Editing and Zinc Finger Nucleases………………………..……..2
1.3. Issues and Limitations………………………………………………………5
1.4.Aims and Objectives………………………………………………..……….6
2. Material and Methods……………………………………………………..8 3. Experiments and Results…………………………………………………11 IV
3.1. Synthesis of FokI-Diamer……………………………………………..……11
3.2.Hierarchical Assembly of Multi-Zinc Finger Arrays……………………..13
3.3.Synthesis of 8ZFs Array…………………………………………………….19
3.4.Synthesis of 8 ZFs in pBM2…………………………………………………23 3.5. Expressino of single chain zinc finger Nuclease…………………………..29
3.6.Conclusions…………………………………………………………..………30
4. PCR Programs……………………………………………………………….31 5. Tables…………………………………………………………………………..34 6. Sequencing Data…………………………………….………………………40 7. References……………………………………………....…………………….69
8. Glossary ………………………………………………………………………71
V
List of Tables 1. Primers 2. Zinc fingers mixtures for Exon-7 Pig GGTA1 3. Zinc fingers mixtures for Exon-9 Pig GGTA1 4. Genomic Targets 5. Some zinc finger arrays in different vectors 6. Nucleotide Sequence of pBM1 Vector 7. Nucleotide Sequence of Linker DNA 8. Nucleotide Sequence of pBM2 9. Nucleotide Sequence of pBM2-SBE7-1 10. Nucleotide Sequence of pBM2-SBE7-2 11. Nucleotide Sequence of pBM2-SBE7-3 12. Nucleotide Sequence of pBM2-SBE7-4 13. Nucleotide Sequence of pBM2-SBE9-1 14. Nucleotide Sequence of pBM2-SBE9-2 15. Nucleotide Sequence of pBM2-SBE9-3 16. Nucleotide Sequence of pBM2-SBE9-4 17. Nucleotide Sequence of pBM2-SBE9-5 18. Nucleotide Sequence of pBM2-SBE9-6 19. Nucleotide Sequence of pBM2-SBE9-7 20. Nucleotide Sequence of pBM2-SBE9-8 VI
21. Protein Sequence of pBM1 Vector 22. Protein Sequence of pc2XB-Linker 23. . Protein Sequence of pBM2 Vector 24. Protein Sequence of pBM2-SBE7-1 25. Protein Sequence of pBM2-SBE7-2 26. Protein Sequence of pBM2-SBE7-3 27. . Protein Sequence of pBM2-SBE7-4 28. 28. Protein Sequence of pBM2-SBE9-1 29. Protein Sequence of pBM2-SBE9-2 30. Protein Sequence of pBM2-SBE9-3 31. Protein Sequence of pBM2-SBE9-4 32. Protein Sequence of pBM2-SBE9-5 33. Protein Sequence of pBM2-SBE9-6 34. Protein Sequence of pBM2-SBE9-7 35. Protein Sequence of pBM2-SBE9-8
VII
List of Figures 1.(a) Schematic representation of typical zinc finger nuclease arrangement (b) Schematic representation of single chain zinc finger dimeric nuclease 2.(a) Scheme of Linking Two FokI catalytic domains with GGGGS Linker and PCR Products of first PCR Reaction. (b) Agarose gel Electrophoresis of PCR Products containing FokI with corresponding Flanking primers (c) 1% Agarose gel Electrophoresis of 10X GGGGS Linker with flanking primers 3.(a) Scheme of Assembly of FokI with Linker in between them with second and third PCR Reaction’s Products (b) 2% Agarose Gel Electrophoresis of FokI assembled with Linker and Linker assembled with FokI (Lane 2 and 3) (c) 2 % Agarose gel electrophoresis showing the PCR product with Two FokI catalytic domains linked via 10xGGGGS linker (d) 2% Agarose gel electrophoresis showing the clone with FokI dimeric PCR Product into pBM2 4. Hierarchical Assembly of Multi-Zinc Finger Arrays by Cut-Ligation-pairwise VIII
5. Electrophoresis analysis. M stands for 100b Marker (Solgent) 1% Agarose gel Electrophoresis of PCR Reaction MM30A1 showing PCR Product of single zinc finger gene 6. 1% Agarose gel Electrophoresis of PCR Reaction MM30A2 showing PCR Product of 2 zinc finger gene 7. 1% Agarose gel Electrophoresis of PCR Reaction MM30B1 showing PCR Product of 4 zinc finger gene. Red arrow shows the 400bp PCR product of 4ZFs 8. 1% Agarose gel Electrophoresis of Colony PCR Reaction MM35C showing PCR Product of colonies which have correct 4 ZFs in pc3XB Vectors. 8 colonies from each of 4 plates containing pc3XB-4ZFs. Red arrows shows 560bp of PCR product of 4ZFs in pc3XB with sequencing primer 9. Cloning of pc3XB-4ZFs vectors to make pc3XB-8ZFs 10. Electrophoresis showing Cloning of pc3XB-4ZFs to make pc3XB-8ZFs 11. 2% Agarose gel Electrophoresis of Colony PCR Reaction MM40D showing PCR Product of colonies which have correct 8 ZFs in pc3XB Vectors (Exon-7). 12. 2% Agarose gel Electrophoresis of Colony PCR Reaction MM40D showing PCR Product of colonies which have correct 8 ZFs in pc3XB Vectors.(Exon-9) IX
13. Scheme of transferring 8ZFs from pc3XB vector into PBM2 Vector 14. Cloning of 8ZFs into pBM2 Vector 15. 2% Agarose gel Electrophoresis of Colony PCR Reaction MM40F showing PCR Product of colonies which have correct 8 ZFs in pBM2 16. (a) Scheme for the Cloning of single chain 4 Zinc finger FokI-Diamer into pTWINI Vector (b) Colony PCR showing the colonies with correct 2.6 kb Insert fragment (c)12% SDS PAGE of IPTG Inducted single chain zinc finger nuclease in E.coli.
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Targeted Genome Editing using Single Chain Zinc Finger Dimeric Nuclease Muhammad Mustafa Department of Chemistry The Graduate School Yonsei University
The ability to achieve site-specific gene manipulation of the genome has widespread implications for basic and applied research. Gene targeting is a process in which site specific mutations are created which lead to gene knockout or an exogenous DNA molecule is incorporated into target site by replacing the corresponding chromosomal segment via homologous recombination to introduce a desired phenotype. Zinc finger genome engineering is based on the introduction of DSB created at a specific locus to activate the cell’s endogenous homologous recombination mechanism with simultaneous supply of exogenous donor DNA to introduce a desired genetic modification. An engineered zinc finger Nucleases (ZFNs) have fusions between the DNA cleavage domain of FokI and a custom-designed ZF binding domain. Two independent ZFNs needs to be organized across the target site on the DNA to create a functional dimeric zinc finger nuclease. Several attempts were made to design rapid assembly methods for the synthesis of custom designed DNA binding domain and to improve the architecture of nuclease for better activity. We developed a Hierarchical Assembly Method for Multi-Zinc Finger Array (HAMZF) to synthesize up to eight zinc finger domains providing 24bp recognition to zinc finger nuclease. XI
The strategy is based on position specific primer design which enables each zinc finger encoding DNA sequence to be arranged according to their relative position. Further the catalytic domains of FokI were linked via flexible (10× GGGGS) amino acids linker and custom designed zinc finger binding domain to synthesize the single chain zinc finger nuclease. The problems related to the PCR based gene synthesis of repeating units were met with the help of specially designed primers and oligos. Several clones containing 4, 6, and 8 zinc fingers were made in pc3XB vector and later transferred to single chain dimeric nuclease to complete the construct. In order to study the expression of these custom made nuclease in prokaryotic system, the genes encoding the single chain zinc finger dimeric nuclease were transferred to pTWINI expression vector downstream of T7 promoter. All the molecular cloning products were analyzed and verified with sequencing.
______________________________________________________________________________ KeyWords: Genome Engineering, Zinc Finger Nuclease, Modular Assembly, FokI Endonuclease,
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1.0
Introduction 1.1.
Zinc Finger Proteins
Zinc finger proteins are most abundant proteins in eukaryotic genomes and have been isolated from various organisms including Yeast, Drosophila melanogaster, Mouse and Humans. They occur as a DNA-binding domain of transcription factors providing DNA sequence specificity. They have diverse functions including DNA recognition, RNA packaging, regulation of apoptosis, transcriptional activation, lipid binding and protein folding and assembly. A zinc finger domain consist of 28-40 amino acids with a characteristic C2H2 (Cys-Cys-HisHis) motif.
It has a simple ββα fold stabilized by hydrophobic interactions supported by
chelation of a single zinc ion. The zinc ion coordinates with cysteine and histidine residues and the order of these coordinating residues are used to classify zinc fingers e.g., Cys2His2, Cys4, and Cys6. Moreover these can be classified into different fold groups based on the overall shape of the protein backbone in the folded domain. The most common or classic zinc finger has fold group termed as Cys2His2 the fold group types are treble clef and zinc ribbon. The first C2H2 zinc finger motif was identified in DNA and RNA binding transcription factor TFIIIA. The sequence specific interactions develops between α-helix domain of zinc finger, which projects itself into the major groove of DNA and specific nucleotides. The base interaction with zinc finger
α-helix depends on the type of zinc finger and nature of
contacts it can make with specific bases. Typically each zinc finger domain recognizes three base pairs of DNA and interestingly simple covalent tandem repeats of zinc finger can recognize longer asymmetric sequences of DNA. 1
The DNA recognition ability of zinc finger triggered a new area of research to synthesis motifs which can recognize literally any sequence of DNA these recognition motifs are then provided with investigative tools to explore the genomic landscape. Majority of zinc finger arrays are based on the zinc finger domain of the murine transcription factor Zif268 or human transcription factor SP1.
.
DNA bound zinc finger protein
1.2. Genome Editing and Zinc Finger Nucleases
The ability to achieve site-specific gene manipulation of the genome has widespread implications for basic and applied research. Gene targeting is a process in which site specific mutations are created which lead to gene knockout or an exogenous DNA molecule is incorporated into target site by replacing the corresponding chromosomal segment via homologous recombination to introduce a desired phenotype.
2
The zinc finger DNA-binding motif exist in many proteins that regulate eukaryotic gene expression. The crystal structure of DNA binded zinc finger protein is resolved and showed that zinc fingers bind in the major groove of DNA and wrap part way around the double helix. Each finger makes its primary contacts in a three-base pair subsite. Residues from the aminoterminal portion of an alpha helix contact the bases, and most of the contacts are made with the guanine-rich strand of the DNA. The C2H2 zinc finger1 proteins are capable of recognizing virtually any DNA sequences2, 3. This property has been successfully utilized for the direct modification of DNA itself via engineered zinc finger nucleases (ZFNs) for gene correction. Zinc Finger Technology successfully manipulate the genomes of higher animals like Drosophila melanogaster, C. elegans, tobacco, corn4, zebrafish 5, various types of mammalian cells 6, and rats
7.
. Clinical
trials for ZF Nuclease that can disrubt the CCR5 gene in CD4+ human T-cells as a potential treatment of HIV/AIDS8. More recently genome editing of mouse model of haemophilia to restore haemostasis20 and hereditable genome editing21 was achieved.
An engineered zinc finger nucleases (ZFNs) have fusions between the DNA cleavage domain of FokI and a custom-designed DNA binding domain. Each Zinc Finger can Recognize three nucleotides sequence of
DNA12. Two Zinc Finger Arrays with FokI catalytic domain can
bind to a specific site with an appropriate spacer to make a Zinc Finger Nuclease13, 14. The length of the spacer between the binding sites of two zinc finger nucleases is an important factor to determine the activity of zinc finger nuclease as well as the site of DSB22.
3
a)
b)
Fig 1. a)Schematic representation of typical zinc finger nuclease arrangement. b) Schematic representation of single chain zinc finger dimeric
nuclease
Zinc finger nucleases opened a new frontier for site-specific gene manipulation with its ability to create a double stranded break (DSB) at position of choice on the genome. A DSB on the genome triggers the cells endogenous homologous recombination machinery and ends 4
up in gene knock out via non-homologous end joining (NHEJ) in the absence of exogenous DNA or in Gene Insertion via Homologous Recombination9, 10 in the presence of exogenous DNA. HR is also used to create regulated genomic rearrangement. In yeast mating type switching during meiosis and generation of immunoglobulin and T-cell receptor diversity in certain species are created by homologous recombination11. In controlled HR, DSB created by a specific nuclease is repaired using a DNA template other than the sister chromatid. Zinc finger based genome engineering is designed to mimic this natural phenomena using DSB created at a specific loci to activate the cell’s endogenous homologous recombination mechanism with simultaneous supply of exogenous donor DNA to introduce a desired genetic modification. The efficiency of the Homologous Recombination is very low in the absence of a position specific DSB and thus is a limiting factor in the world of Targeted Genome Editing. Better activity of the ZFNs will increase the efficiency of Homologous Recombination and increases specificity of ZFNs will reduce the cytotoxicity and off site DNA damage. Any technique which can improve the activity and specificity of the ZFNs will provide access to
the un-
explored genomic targets of complex organisms.
1.3. Issues and Limitations With all its efficient and fancy design and strategy zinc finger based nucleases practically has many limitations need to be worked on.
5
1. Availability of target site for zinc finger recognition motifs on sense and antisense strand of the DNA at a proper spacer length to create a functional dimeric FokI endonuclease. 2. Individual delivery of each of the component, ZFR and ZFL of zinc finger nuclease to target DNA (Fig 1a) 3. Maintenance of minimum affinity threshold between zinc finger proteins and DNA to allow a successful DSB to avoid cellular cytotoxicity 15
Some alternate approaches to design new model for the zinc finger nuclease to reduce the limitations was already done. Two groups have engineered the FokI-dimer interface so that the engineered ZFN preferentially formed a heterodimer16, 17 and Sandwiched zinc finger nuclease18. A similar approach with 12 bp recognition was reported for mitochondrial DNA target site19
1.4 Aims and Objective In order to improve the design of zinc finger nuclease and to make a more active and specific zinc finger nuclease we design and synthesize the Sing Chain Dimeric zinc finger nuclease. The single chain dimeric zinc finger nuclease has 24bp recognition site with FokI –Dimeric endonuclease linked with flexible amino acid linker (Fig 1b). The said nuclease can be delivered with single donor plasmid to cell and its expression could produce a nuclease 6
capable of effectively recognize and cleave the target site. The synthetic approach that was selected to synthesized these products was based on PCR methods in order to reduce the time and cost of synthesis. The biggest challenge while working with these types of gene products was the existence of large number of repeating units within the single PCR product. Such situations reduces the efficiency and yield and the problems were met using primer based synthesis.
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2.0 Materials and Methods Catalytic domains of FokI endonuclease was amplified from pBM1 Vector (Table 6 and Table 21’)
using the Flanking Primers designed for Assembly of FokI-Dimer . 10X
GGGGS Linker was synthesized in pc3XB vector (‘addgene’) and amplified as well with custom designed flanking primers (Fig 2, Table 7 and Table 22). The amplicons were assembled in two stage PCR Reaction to synthesize FokI-Dimer (Fig 3, Table 8 and Table 23) 4 genomic targets for Gene manipulations were selected (Table4). The Modular Assembly for the Synthesis of 4ZFs
started from kits of plasmids ‘‘Zinc Finger Consortium Modular
Assembly Kit v1.0’’ which consists of pc3XB vector-based 141 different zinc finger modules. Every zinc finger modules have the same restriction enzyme sites of XbaI, XmaI, AgeI and BamHI both ends of zinc finger genes. (Fig 4) Zinc finer corresponding to the target sites with respect to their position were amplified, purified and mixed to make a library with 50 ng of each of the ZF DNA Fragment (Table 2 and Table 3) because different ZFs are developed targeting single nucleotide triplet. Two sets of primers (set1 primers(blue) and set2 primers(red)) were designed which have two different annealing regions. One part of the primers anneals(black line) to both end of the zinc finger module in pc3XB vector with 50 ºC of Tm and another part of the primers(blue or red line) doesn’t anneal to it. And two sets of primers have different sequences of annealing sites to each other. PCR amplification was done for each zinc finger module using these primers with annealing temperature of 50 ºC. We amplified F1 and F4 modules using ‘set1 primers (blue) and F2 and 8
F3 using ‘set2 primers (red). PCR products with one ZF were analyzed on 1% Agarose gel and PCR products w of 140bp size were removed and purified from the gel
(Fig 4 and Fig
5) Restriction enzyme digestion was performed using AgeI for F1 and F3, XmaI for F2 and F4. AgeI and XmaI have different recognition site but have same overhangs after digestion. Even though F1 and F3 are cleaved using AgeI and F2 and F4 are cleaved using XmaI, F1 and F2, F3 and F4 can be ligated because the overhang sequences are same. F1 and F2, F3 and F4 were ligated
with T4 DNA ligase and used
2 ZFs (Fig 4 and Fig 6).For F1-F2, primer(red)’ were used
and for F3-F4
as templates for PCR amplification of
‘set1 forward primer(blue)’and ‘set2 reverse ‘set2 forward primer(red) and ‘set1 reverse
primer(blue) were used with68 ºC. The restriction and ligation cycle was repeat again to make 4ZFs (Fig 4 and Fig 7) Ligated 4ZFs product acted as a template with‘set1 forward primer and reverse primer (blue)’ to give 4ZFs PCR product which was later Cloned into pc3XB Vector (Fig 4 and Fig 8) The 8 zinc finger construct was made in two steps. First the 4 zinc finger construct was made using modular assembly and transferred to pc3XB vector using the XbaI and BamHI enzyme sites. These two sets of 4ZFs were later joined to form 8 zinc finger array using the AgeI,XmaI and HindiIII (Fig 8 and Table 4) restriction sites. The 8 zinc finger array from pc3XB vector was transferred to pBM2 Vector using the XbaI and BamHI sites to complete the pBM2-8ZFs (Fig 13). The sequences of the correct clones containing single chain dimeric nuclease are give in Table 9-21. Their corresponding protein sequences were mentioned in Table 21-35 Two additional constructs with 6ZFs array targeting galK gene and 4ZFs array targeting narK gene on the E.coli
genomes were also constructed in the same way. For
Expression analysis of gene containing 8 zinc finger single chain Dimeric nuclease the DNA 9
encoding 8 zinc finger and single chain FokI-Dimeric nuclease were transferred to pTWINI vector with NdeI and AscI site (Fig ? Table ?). The NdeI site was incorporated using primers and insert was cloned downstream of T7 promoter of pTWINI vector. The cell extracts after induction with IPTG were analyzed on 12% SDS PAGE.
Zinc finger encoding plasmids were purchased from ;addgene’. PCR Reactions were carried out in BIORAD C1000TM Thermal Cycler using 1 µl of 10 µM Forward and Reverse Primers and 1 µl of Template DNA using 10 µl
pfu-polymerase Premix (Solgent) in 20 µl PCR
Reaction Until describe otherwise. Colony PCR Reactions were carried out in the same way with an exception of pfu-polymerase (Solgent) instead Taq-Polymerase Premix (Solgent) were used. PCR Products were purified from the Gel using ‘BIONEER Gel purification Kit’ and Restriction enzymes and other impurities were removed from the DNA using ‘BIONEER PCR purification Kit’ Plasmids from the correct clones were extracted using ‘BIONEER Plasmid Extraction Kit’. Most of the Cloning techniques and procedure were carried out using standard Molecular Biology protocols. The Plasmid DNA were sequenced from ‘Macrogen’ using standard Sanger Sequencing.
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3.0.
Experiments and Results
3.1. Synthesis of Single Chain FokI-Dimer
a
b
Fig 2
c
a) Scheme of Linking Two FokI catalytic domains with GGGGS Linker and PCR Products of first PCR Reaction. b) 2% Agarose gel Electrophoresis of PCR Products containing FokI with corresponding Flanking
primers. c) 1% Agarose gel
Electrophoresis of 10X GGGGS Linker with flanking primers
11
a
c
d
b
Fig 3. a) Scheme of Assembly of FokI with Linker in between them with second and third PCR Reaction’s Products. b) 2% Agarose Gel Electrophoresis of FokI assembled with Linker and Linker assembled with FokI (Lane 2 and 3). c) 2 % Agarose gel electrophoresis showing the PCR product with Two FokI catalytic domains linked via 10xGGGGS linker. d) 2% Agarose gel electrophoresis showing the clone with FokI dimeric PCR Product into pBM2.
12
3.2 Hierarchical Assembly of Multi-Zinc Finger Arrays
Fig 4. Hierarchical Assembly of Multi-Zinc Finger Arrays by Cut-Ligation-pairwise PCR Method. F1,F2,F3 and F4 are defined ZFs according to their position on the target site. The final 4ZFs PCR product was cloned into pc3XB vector using XbaI and BamHI restriction enzyme sites
13
M 7A1 7A2 7A3 7A4
ZF Target position Codon 7A1
1
AAG
7A2
2
GAA
7A3
3
AAC
7A4
4
GGC
7B1
5
ATA
7B2
6
GCT
7B3
7
GAC
7B4
8
GAA
M
M 7B1 7B2 7B3 7B4
M
M 9A1 9A2 9A3 9A4
M
ZF Target position Codon 9A1
1
AAG
9A2
2
GAA
9A3
3
AAC
9A4
4
GGC
9B1
5
ATA
9B2
6
GCT
9B3
7
GAC
9B4
8
GAA
M 9B1 9B2 9B3 9B4 M
14
Fig 5 Electrophoresis analysis. M stands for 100b Marker (Solgent) 1% Agarose gel Electrophoresis of PCR Reaction MM30A1 showing PCR Product of single zinc finger gene.
ZF position
Target Codon
7A1-2
2-1
GAAAAG
7A3-4
4-3
GGCAAC
7B1-2
6-5
GCTATA
7B3-4
8-7
GAAGAC
ZF position
Target Codon
9A1-2
2-1
CAAGGC
9A3-4
4-3
GTGGTA
9B1-2
6-5
GGCCTG
9B3-4
8-7
GCTACA
M
7 A1-2 7A3-4 7B1-2 7B3-4
M 9A1-2 9A3-4 9B1-2 9B3-4
M
M
Fig 6. 1% Agarose gel Electrophoresis of PCR Reaction MM30A2 showing PCR Product of 2 zinc finger gene. 15
ZF position
Target Codon
7A
4-3-2-1
GGCAACGAAAAG
7B
8-7-6-5
GAAGAC GCTACA
M
ZF Target Codon position 9A
4-3-2-1
GTGGTACAAGGC
9B
8-7-6-5
GCTACAGGCCTG
M
7A
9A
7A
9A
7B
9B
7B
9B
M
M
Fig7. 1% Agarose gel Electrophoresis of PCR Reaction MM30B1 showing PCR Product of 4 zinc finger gene. Red arrow shows the 400bp PCR product of 4ZFs
16
Colony-ID 1A
pc3XB-7A-1A-MM
1B
pc3XB-7A-1B-MM
1C
pc3XB-7A-1C-MM
1D
pc3XB-7A-1D-MM
1E
pc3XB-7A-1E-MM
1F
pc3XB-7A-1F-MM
1G
pc3XB-7A-1G-MM
1H
pc3XB-7A-1H-MM
2A
pc3XB-7B-2A-MM
2B
pc3XB-7B-2B-MM
2C
pc3XB-7B-2C-MM
2D
pc3XB-7B-2D-MM
2E
pc3XB-7B-2E-MM
2F
pc3XB-7B-2F-MM
2G
pc3XB-7B-2G-MM
2H
pc3XB-7B-2H-MM
M 1A 1B 1C 1D 1E 1F 1G 1H M 2A 2B 2C 2D 2E 2F 2G 2H M
17
Colony-ID 1A
pc3XB-9A-1A-MM
1B
pc3XB-9A-1B-MM
1C
pc3XB-9A-1C-MM
1D
pc3XB-9A-1D-MM
1E
pc3XB-9A-1E-MM
1F
pc3XB-9A-1F-MM
1G
pc3XB-9A-1G-MM
1H
pc3XB-9A-1H-MM
2A
pc3XB-9B-2A-MM
2B
pc3XB-9B-2B-MM
2C
pc3XB-9B-2C-MM
2D
pc3XB-9B-2D-MM
2E
pc3XB-9B-2E-MM
2F
pc3XB-9B-2F-MM
2G
pc3XB-9B-2G-MM
2H
pc3XB-9B-2H-MM
M 1A 1B 1C 1D 1E 1F 1G 1H M 2A 2B 2C 2D 2E 2F 2G 2H M
Fig 8. 1% Agarose gel Electrophoresis of Colony PCR Reaction MM35C showing PCR Product of colonies which have correct 4 ZFs in pc3XB Vectors. 8 colonies from each of 4 plates containing pc3XB-4ZFs. Red arrows shows 560bp of PCR product of 4ZFs in pc3XB with sequencing primer.
18
3.3. Synthesis of 8ZFs Array
Fig 9. Cloning of pc3XB-4ZFs vectors to make pc3XB-8ZFs.
19
M
100bp Marker M
1
pc3XB-7A-Mix-PE
2
pc3XB-7B-Mix-PE
3
7B-GP
4
pc3XB-9A-Mix-PE
5
pc3XB-9B-Mix-PE
6
9B-GP
M
100bp Marker
1
2
3
4
5
6
M
Fig 10. Electrophoresis showing Cloning of pc3XB-4ZFs to make pc3XB-8ZFs. Lane 1 and 4 (digested with AgeI/HindiIII) will provide the vector backbone with 4 ZFs already present. Lane 2,3 and 5,6 (digested with XmaI/HindiIII)give the 4ZFs Insert fragment. Red arrow indicating the 4ZFs digested fragments(Lane 2,5) and PCR product (Lane 3,6) of 400bp
20
M
1kb Marker
1A
pc3XB-7AB-1A-MMA
1B
pc3XB-7AB-1B-MMA
1C
pc3XB-7AB-1C-MMA
1D
pc3XB-7AB-1D-MMA
1E
pc3XB-7AB-1E-MMA
1F
pc3XB-7AB-1F-MMA
1G
pc3XB-7AB-1G-MMA
1H
pc3XB-7AB-1H-MMA
2A
pc3XB-7AB-2A-MMA
2B
pc3XB-7AB-2B-MMA
2C
pc3XB-7AB-2C-MMA
2D
pc3XB-7AB-2D-MMA
2E
pc3XB-7AB-2E-MMA
2F
pc3XB-7AB-2F-MMA
2G
pc3XB-7AB-2G-MMA
2H
pc3XB-7AB-2H-MMA
Fig 11.
M 1A 1B 1C 1D
1E 1F 1G 1H M 2A 2B 2C 2D 2E 2F 2G 2H M Mix
2% Agarose gel Electrophoresis of Colony PCR Reaction MM40D showing PCR Product of colonies which have correct 8 ZFs in pc3XB Vectors (Exon-7). 16 colonies from the plate containing pc3XB-8ZFs library targeting Exon 7 of pig GGTA1were analyzed.Red arrow with the correct clone size of 895bp.
21
M
1kb Marker
1A
pc3XB-9AB-1A-MMA
1B
pc3XB-9AB-1B-MMA
1C
pc3XB-9AB-1C-MMA
1D
pc3XB-9AB-1D-MMA
1E
pc3XB-9AB-1E-MMA
1F
pc3XB-9AB-1F-MMA
1G
pc3XB-9AB-1G-MMA
1H
pc3XB-9AB-1H-MMA
2A
pc3XB-9AB-2A-MMA
2B
pc3XB-9AB-2B-MMA
2C
pc3XB-9AB-2C-MMA
2D
pc3XB-9AB-2D-MMA
2E
pc3XB-9AB-2E-MMA
2F
pc3XB-9AB-2F-MMA
2G
pc3XB-9AB-2G-MMA
2H
pc3XB-9AB-2H-MMA
M 1A 1B 1C 1D 1E 1F 1G 1H
Fig12.
M 2A 2B 2C 2D 2E 2F 2G 2H M Mix
2% Agarose gel Electrophoresis of Colony PCR Reaction MM40D showing PCR Product of colonies which have correct 8 ZFs in pc3XB Vectors.(Exon-9) 16 colonies from the plate containing pc3XB-8ZFs library targeting Exon 9 of pig GGTA1were analyzed. Red arrow with the correct clone size of 895bp. 22
3.4.Synthesis of 8 ZFs in pBM2
Fig 13. Scheme of transferring 8ZFs from pc3XB vector into PBM2 Vector
23
M pBM2 pBM2-R 2C
1kb Marker pBM2 (Un-cut) pBM2-Digested pc3XB-Ex7-7AB-2C
2F 3G 4A M
pc3XB-Ex7-7AB-2F pc3XB-Ex7-7AB-3G pc3XB-Ex7-7AB-4A 1kb Marker
M pBM2 pBM2-R 2C 2F
Fig 14.
3G
4A M
M pBM2 pBM2-R 4B 4B2 5C 6A M
1kb Marker pBM2 (Uncut) pBM2-Digested pc3XB-Ex9-9AB-4B pc3XB-Ex9-9AB-4B2 pc3XB-Ex9-9AB-5C pc3XB-Ex9-9AB-6A 1kb Marker
M pBM2pBM2-R 4B
4B2 5C
6A
M
Cloning of 8ZFs into pBM2 Vector. pBM2 vector as well as 4 colonies for each target site containing pc3XB-8ZFs targeting Exon 7 and Exon 9 of pig GGTA1 were digested with XbaI and BamHI restriction enzyme to get pBM2 vector backbone and 8ZFs DNA fragment. Red arrow indicates the 670bp fragment of 8ZFs. Sample 2C did not give any 8ZFs fragment.
24
M
1kb Marker
1A
pBM2-7-2C-1A-MM 2B pBM2-7-2F-2B-MM
1B
pBM2-7-2C-1B-MM 2C pBM2-7-2F-2C-MM
1C
pBM2-7-2C-1C-MM 2D pBM2-7-2F-2D-MM
1D
pBM2-7-2C-1D-MM 2E
pBM2-7-2F-2E-MM
1E
pBM2-7-2C-1E-MM
2F
pBM2-7-2F-2F-MM
1F
pBM2-7-2C-1F-MM
2G pBM2-7-2F-2G-MM
1G
pBM2-7-2C-1G-MM 2H pBM2-7-2F-2H-MM
1H
pBM2-7-2C-1H-MM M
C
Control
M 1A 1B 1C 1D 1E 1F 1G 1H
Fig 15.
2A pBM2-7-2F-2A-MM
1kb Marker
C 2A 2B 2C 2D 2E 2F 2G 2H M
2% Agarose gel Electrophoresis of Colony PCR Reaction MM40F showing PCR Product of colonies which have correct 8 ZFs in pBM2 Vectors. 8 colonies were
25
picked from 8 plates(4 for each target) containing pBM2-8ZFs clones and analyzed with sequencing primers. Red arrow indicating the correct clones with 1300bp size. M
1kb Marker
2A pBM2-7-4A-2A-MM
1A pBM2-7-3G-1A-MM 2B pBM2-7-4A-2B-MM 1B pBM2-7-3G-1B-MM 2C pBM2-7-4A-2C-MM 1C pBM2-7-3G-1C-MM 2D pBM2-7-4A-2D-MM 1D pBM2-7-3G-1D-MM 2E
pBM2-7-4A-2E-MM
1E
pBM2-7-3G-1E-MM
2F
pBM2-7-4A-2F-MM
1F
pBM2-7-3G-1F-MM
2G pBM2-7-4A-2G-MM
1G pBM2-7-3G-1G-MM 2H pBM2-7-4A-2H-MM 1H pBM2-7-3G-1H-MM M C
1kb Marker
Control
M 1A 1B 1C 1D 1E 1F 1G 1H C 2A
2B 2C 2D 2E 2F 2G 2H M
26
Fig 15 continued M
1kb Marker
2A pBM2-9-4B2-2A-MM
1A pBM2-9-4B-1A-MM 2B pBM2-9-4B2-2B-MM 1B pBM2-9-4B-1B-MM 2C pBM2-9-4B2-2C-MM 1C pBM2-9-4B-1C-MM 2D pBM2-9-4B2-2D-MM 1D pBM2-9-4B-1D-MM 2E
pBM2-9-4B2-2E-MM
1E
pBM2-9-4B-1E-MM
2F
pBM2-9-4B2-2F-MM
1F
pBM2-9-4B-1F-MM
2G pBM2-9-4B2-2G-MM
1G pBM2-9-4B-1G-MM 2H pBM2-9-4B2-2H-MM 1H pBM2-9-4B-1H-MM M C
1kb Marker
Control
M
1A 1B 1C
1D 1E 1F 1G 1H C 2A 2B 2C 2D 2E
Fig 15 continued 27
2F 2G 2H
M
M
1kb Marker
2A pBM2-9-6A-2A-MM
1A pBM2-9-5C-1A-MM 2B pBM2-9-6A-2B-MM 1B pBM2-9-5C-1B-MM 2C pBM2-9-6A-2C-MM 1C pBM2-9-5C-1C-MM 2D pBM2-9-6A-2D-MM 1D pBM2-9-5C-1D-MM 2E
pBM2-9-6A-2E-MM
1E
pBM2-9-5C-1E-MM
2F
pBM2-9-6A-2F-MM
1F
pBM2-9-5C-1F-MM
2G pBM2-9-6A-2G-MM
1G pBM2-9-5C-1G-MM 2H pBM2-9-6A-2H-MM 1H pBM2-9-5C-1H-MM M C
1kb Marker
Control
M 1A 1B 1C 1D 1E 1F
1G 1H C
2A 2B 2C 2D
Fig 15 continued 28
2E 2F 2G 2H M
Expression of single chain zinc finger Nuclease (a)
(b) M
M
1kb Marker
1B
pTWINI-8ZFs-FokI-D
1A 1B 1C 1D 1E
1F
1G 1H M
(c) M
M
Marker (Fermentas Prestained Protein Ladder)
A
Negative Control
B
pTWINI-8ZFs-FokI-D-1
C
pTWINI-8ZFs-FokI-D-2
M
Marker (Fermentas Prestained Protein Ladder)
Fig.16
A
B
C
M
(a) Scheme for the Cloning of single chain 4 Zinc finger FokI-Dimer into pTWINI Vector. (b) Colony PCR showing the colonies with correct 2.6 kb Insert fragment. (c)12% SDS PAGE of IPTG Inducted single chain zinc finger nuclease in E.coli. Red arrow showing the 89.2KDa target size 29
Conclusion The Genes which can encode single chain 8 and 4 zinc finger array fused with Two FokI catalytic domains Targeting different target sites (Table 4) were synthesized successfully. The integrity was established using sequencing and these vector are now in Use for In-Vivo expression and gene targeting in their corresponding hosts. For the Expression analysis in E.coli the gene for single chain zinc finger dimeric nuclease was transferred to pTWINI vector downstream to T7 promoter. The synthetic problems related to the amplification of larger genes with repeating sequences were overcome by using specially designed flanking primers with optimized PCR conditions. The Molecular cloning showed that a 24bp recognition domain motif using 8 zinc finger array can be assembled efficiently. The problems associated with off-site activity and cytotoxicity will be decreased while working with genome editing of mammalian cell lines. PCR based synthesis of single chain dimeric nuclease can be used to assemble FokI-Dimer with varying lengths of linker fragment. One observation about the nature of flexible linker required further investigation which is that its flexibility might reduce the specificity of the cleavage and rigid linker in that cased would be a better choice.
30
4.0. PCR Programs Program Name MM30A1 Expected Size 148bp (for lane 2,5) 124bp (for lane3,4) 1. Initial Heating 95 °C 3.00 min 2. Denaturing
95 °C
0.30 min
3. Annealing
55 °C
0.30
72 °C
0.15 min
4. Elongation 5. Repeat 24 6. Final
min
30 cycles
Elongation
7. Incubation
72 °C
10.00 min
4 °C
∞
Program Name MM30A2 Expected Size 232bp 1. Initial Heating
95 °C
3.00 min
2. Denaturing
95 °C
0.30 min
3. Annealing
68 °C
0.30
72 °C
0.20 min
4. Elongation 5. Repeat 24 6. Final
min
30 cycles
Elongation
7. Incubation
31
72 °C
10.00 min
4 °C
∞
Program Name MM30B1 Expected Size 400bp 1. Initial Heating
95 °C
3.00 min
2. Denaturing
95 °C
0.30 min
3. Annealing
55 °C
0.30
72 °C
0.30 min
4. Elongation 5. Repeat 24 6. Final
min
30 cycles
Elongation
7. Incubation
72 °C
10.00 min
4 °C
∞
Program Name MM35C Expected Size 559bp 1. Initial Heating
95 °C
3.00 min
2. Denaturing
95 °C
0.30 min
3. Annealing
60 °C
0.30
72 °C
0.30 min
4. Elongation 5. Repeat 24 6. Final
min
35 cycles
Elongation
7. Incubation
32
72 °C
10.00 min
4 °C
∞
Program Name MM40D Expected Size 895bp 1. Initial Heating
95 °C
3.00 min
2. Denaturing
95 °C
0.30 min
3. Annealing
60 °C
0.30
72 °C
1.00 min
4. Elongation 5. Repeat 24 6. Final
min
40 cycles
Elongation
7. Incubation
72 °C
10.00 min
4 °C
∞
Program Name MM40F Expected Size 1300bp 1. Initial Heating
95 °C
3.00 min
2. Denaturing
95 °C
0.30 min
3. Annealing
60 °C
0.30
72 °C
1.30 min
4. Elongation 5. Repeat 24 6. Final
min
40 cycles
Elongation
7. Incubation
33
72 °C
10.00 min
4 °C
∞
5.0. Tables Table 1 Primers Primer
5 Sequencing
KHB090807 ZF For Pri
CTAACTAGAGAACCCACTGCTTAC
pc3XB Seq Rev
GCACCTTCCAGGGTCAAG
CMV_fwd_primer
CGCAAATGGGCGGTAGGCGTG
BGH_rev
TAGAAGGCACAGTCGAGG
F1
CGCAAATGGGCGGTAGGCGTG
R1
CGATGCAGTAAGCTGGACAAAGTTTATCTCGCCGTTATT AAAT
F2
GTCCAGCTTACTGCATCGCCCGGGGGCGGCGG
R2
TACGACCGATCGCAATACACCACCACCGCCTGAG
F3
GTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACT GGAG
R3
CAGATGGCTGGCAACTAGAAG
pTWINI-Seq-For
TAATACGACTCACTATAGGG
pTWINI-Seq-Rev
TATGCTAGTTATTGCTCAG
34
Table 2. Zinc fingers mixtures for Exon-7 Pig GGTA1 Exon-7 Target Site:
GAA GAC GCT ATA GGC AAC GAA AAG
ZF Position
Target Codon
8
GAA
7
6
5
GAC
GCT
ATA
ZF ID
Plasmid Conc. (ng/µl)
Product conc. (ng/µl )
V(µl) for 50 ng DNA
1
17.3
15.1
3.311258
18
168.4
11.3
4.424779
36
141.8
18.7
2.673797
63
250.4
19.2
2.604167
116
87.6
15.9
3.144654
122
86.9
23.7
2.109705
123
64.4
16.1
3.10559
124
88.6
15.4
3.246753
2
120.4
22.9
2.183406
19
81.7
27.1
1.845018
37
137.1
26.9
1.858736
65
211
20.2
2.475248
107
22.3
23.3
2.145923
110
95.7
24.4
2.04918
8
138.5
23.8
2.10084
24
124.4
8.1
6.17284
43
153.8
27.3
1.831502
72
232.4
23.4
2.136752
139
108.3
26.1
1.915709
86
64.1
25
2
35
4
GGC
10
141.4
19.5
2.564103
26
240.4
17.4
2.873563
45
87.5
20.5
2.439024
61
170
24.2
2.066116
3
AAC
75
105.8
18.2
2.747253
2
GAA
1
17.3
15.1
3.311258
18
168.4
11.3
4.424779
36
141.8
18.7
2.673797
63
250.4
19.2
2.604167
116
87.6
15.9
3.144654
122
86.9
23.7
2.109705
123
64.4
16.1
3.10559
124
88.6
15.4
3.246753
18.8
2.659574
1
AAG
76 344/2
36
Table 3. Zinc fingers mixtures for Exon-9 Pig GGTA1 Exon-9 Target: GCT ACA GGC CTG GTG GTA CAA GGC ZF Position
Target Codon
8
GCT
ZF ID
Plasmid Conc. (ng/µl)
Product conc. (ng/µl )
V(µl) for 50 ng DNA
8
138.5
23.8
2.10084
24
124.4
8.1
6.17284
43
153.8
27.3
1.831502
72
232.4
23.4
2.136752
139
108.3
26.1
1.915709
7
ACA
78
243.2
26.5
1.886792
6
GGC
10
141.4
19.5
2.564103
26
240.4
17.4
2.873563
45
87.5
20.5
2.439024
61
170
24.2
2.066116
55
168
30.7
1.628664
22.8
2.192982
5
CTG
102 730.4/5 4
3
GTG
GTA
15
258.5
24.1
2.074689
31
182.5
24
2.083333
49
80.2
28.9
1.730104
66
196.4
23.5
2.12766
138
149.4
22.9
2.183406
13
139.1
30.7
1.628664
29
83.3
30.8
1.623377
47
200.4
28.3
1.766784
37
2
1
CAA
GGC
67
258.9
29.5
1.694915
126
103.2
29.7
1.683502
127
170.1
28.4
1.760563
89
258.6
25.9
1.930502
121
207.6
26.9
1.858736
125
179.3
23.2
2.155172
10
141.4
19.5
2.564103
26
240.4
17.4
2.873563
45
87.5
20.5
2.439024
61
170
24.2
2.066116
Table 4. Genomic Targets Target Gene
Sequence
Exon7 pig GGTA1
5 GAA GAC GCT ATA GGC AAC GAA AAG
Exon 9 pig GGTA1
GCT ACA GGC CTG GTG GTA CAA GGC
narK E.coli
GAA GAC GCT ATA
galK E.coli
5 GTC GTT GTA GTC GGT GTG
38
Table 5. Some zinc finger arrays in different vectors Sequence of Zinc fingers in different vectors produced Sample
Vector with ZFs ID
pc3XB-7A-1E
pc3XB-76-116-76-26
pc3XB-7B-2A
pc3XB-86-139-65-123
pc3XB-9A-1C
pc3XB -26-121-127-138
pc3XB-9B-2H
pc3XB -102-26-78-8
pc3XB-7AB-2F-MM
pc3XB-52-116-75-10-86-139-107-17
pc3XB-9AB-2B-MM
pc3XB-10-125-127-15-102-26-78-139
pBM2-7-2F-2D-MM
pBM2-52-116-75-10-86-139-107-17
pBM2-7-3G-1B-MM
pBM2-52-116-75-45-86-139-107-116
pBM2-7-4A-2A-MM
pBM2-52-123-75-10-86-139-107-116
pBM2-9-4B-1A-MM
pBM2-10-125-127-15-102-26-78-139
pBM2-9-4B2-2H-MM
pBM2-66-89-29-138-55-26-78-139
pBM2-9-5C-1G-MM
pBM2-26-121-29-138-102-26-78-139
pBM2-9-6A-2D-MM
pBM2-10-125-127-15-55-26-78-139
39
Table.6. Nucleotide Sequence of pBM1 Vector ATGACGACGATAAATCTAGATAGTAAGCTTGGATCCCAACTAGTCAAA AGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATAT GTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTC AGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTA TGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGC AATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACT AAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAA ATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAAC CCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGT TTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTAC ACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAA GAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAG AGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTT GGAGGTG GCAGCGGTGGCGGTGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACG TCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Bold
XbaI and BamHI restriction enzyme sites respectively
Under Line Black
FokI- Catalytic Domain
Table.7. Nucleotide Sequence of Linker DNA TCTAGACCCGGGGTCCAGCTTACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCG GTGGTTCAGGTGGAGGCGGTTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTG GCGGTGGTTCAGGCGGTGGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCT GGTGGCGGTGGCTCAGGCGGTGGTGGTGTATTGCGATCGGTCGTAACCGGTGGAT CC Italic
Linker DNA sequence
40
Table.8. Nucleotide Sequence of pBM2 ATGACGACGATAAATCTAGATAGTAAGCTTGGATCCCAACTAGTCAAAAGTGA ACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGA ATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAA ATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGG GTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTA CGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGC CAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACA TATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAG TTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGAT TAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAAT TGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAA ATTTAATAACGGCGAGATAAACTTTGTcCAgCTTACTgcATCGCCCGGGGGCGGCGG TTCTGGTGGCGGTGGTTCAGGTGGAGGCGGTTCTGGTGGTGGCGGTTCAGGTGGTG GTGGTTCAGGTGGCGGTGGTTCAGGCGGTGGTGGTTCTGGCGGAGGCGGTTCAGGT GGCGGTGGTTCTGGTGGCGGTGGCTCAGGCGGTGGTGGTgtaTTGCGATCGGTCGTA CAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTG AAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTC AGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATA TAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGT CGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGT TATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAAT CAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCT TCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACA AAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAG TGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTA GAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGGAGGTGGCAGC GGTGGCGGTGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAAT CCTGGCCCATTCGCCGGCGCATAG
Bold
XbaI and BamHI restriction enzyme sites respectively
Under Line Black
FokI- Catalytic Domain
Italic
Linker
41
Table.9. Nucleotide Sequence of pBM2-SBE7-1 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGAAATTTGCCTGCCCTGAGTGTCCTAAGCG CTTCATGCGTAGCGACAACCTGACACAGCATATCAAGACCCACACCGGGGAGAA GCCTTTTCAATGCAACCAGTGCGGTGCGAGCTTCACGCAAAAGGGCAACTTACTG CGCCACATCAAGCTGCATACCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGA AAGAGTTTTAGCGATTCTGGAAATCTCAGAGTGCACCAGAGAACACATACCGGG GAGAAGAAATTTGCCTGCCCTGAGTGTCCTAAGCGCTTCATGGATCGTAGCCACC TCGCACGTCATATCAAGACCCACACCGGGGAGAAGCCTTACAAATGCCCAGAAT GTGGAAAGAGTTTTAGCCAAAAGTCTTCCCTCATCGCTCACCAGAGAACACATAC CGGGGAGAAGCCTTACGAGTGCAACTACTGCGGCAAGACGTTCAGCGTGAGCTC CACGCTCATTCGCCACCAGCGCATCCACACCGGGGAGAAGCCTTACAAATGCAA GCAGTGCGGCAAGGCTTTCGGATGTCCCAGCAATCTGAGACGGCACGGGCGGAC CCATACCGGGGAGAAGCCGCATATTTGCCACATCCAAGGCTGTGGGAAGGTGTA CGGCCAACGCAGCAACCTGGTACGTCACTTGCGCTGGCATACCGGTGGATCCCA ACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAA ATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAG GATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATA GAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCG GATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTA TAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCA AACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCT GTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAG CTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGT AGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGA GGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCAGCTTACTGC ATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGGCGGTTCTGG TGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGCGGTGGTGGT TCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCTCAGGCGGT GGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGGAGGAGAAG AAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAA TTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGG AATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAA ACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTG GATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAA ATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAAT GAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGA GTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCAC TAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATG 42
ATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGC GAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGAAGTCTGCTA ACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Sample ID
pBM2-SBE7-1
ZFs
76-116-75-10-86-139-37-116
Target DNA
GAA GAC GCT ATA GGC AAC GAA AAG
Table.10. Nucleotide Sequence of pBM2-SBE7-2 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGAAATTTGCCTGCCCTGAGTGTCCTAAGCG CTTCATGCGTAGCGACAACCTGACACAGCATATCAAGACCCACACCGGGGAGAA GCCTTTTCAATGCAACCAGTGCGGTGCGAGCTTCACGCAAAAGGGCAACTTACTG CGCCACATCAAGCTGCATACCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGA AAGAGTTTTAGCGATTCTGGAAATCTCAGAGTGCACCAGAGAACACATACCGGG GAGAAGCCGCATATTTGCCACATCCAAGGCTGTGGGAAGGTGTACGGCGATCGT AGCCACCTGACACGTCACTTGCGCTGGCATACCGGGGAGAAGCCTTACAAATGC CCAGAATGTGGAAAGAGTTTTAGCCAAAAGTCTTCCCTCATCGCTCACCAGAGA ACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGCGGCAAGACGTTCAGC GTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACCGGGGAGAAGCCTTAC AAATGCAAGCAGTGCGGCAAGGCTTTCGGATGTCCCAGCAATCTGAGACGGCAC GGGCGGACCCATACCGGGGAGAAGCCGCATATTTGCCACATCCAAGGCTGTGGG AAGGTGTACGGCAAGAACTGGAAGCTCCAGGCGCACTTGCGCTGGCATACCGGT GGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCAT AAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATT CCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTA TGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTA TACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGC GGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAA GAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTAT CCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAA CTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTT CTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAA CCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCAGC TTACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGGCG 43
GTTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGCGG TGGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCTCA GGCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGGAG GAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTG AATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGT AATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCA AGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGA TCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAG ATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACC CTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTT GTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCAT ATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAG AAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATA ACGGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGAAGT CTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Sample ID
pBM2-SBE7-2
ZFs
52-116-75-45-86-139-107-1
Target DNA
GAA GAC GCT ATA GGC AAC GAA AAG
Table.11. Nucleotide Sequence of pBM2-SBE7-3 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGAAATTTGCCTGCCCTGAGTGTCCTAAGCG CTTCATGCGTAGCGACAACCTGACACAGCATATCAAGACCCACACCGGGGAGAA GCCTTTTCAATGCAACCAGTGCGGTGCGAGCTTCACGCAAAAGGGCAACTTACTG CGCCACATCAAGCTGCATACCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGA AAGAGTTTTAGCGATTCTGGAAATCTCAGAGTGCACCAGAGAACACATACCGGG GAGAAGCCGCATATTTGCCACATCCAAGGCTGTGGGAAGGTGTACGGCGATCGT AGCCACCTGACACGTCACTTGCGCTGGCATACCGGGGAGAAGCCTTACAAATGC CCAGAATGTGGAAAGAGTTTTAGCCAAAAGTCTTCCCTCATCGCTCACCAGAGA ACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGCGGCAAGACGTTCAGC GTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACCGGGGAGAAGCCGCAT ATTTGCCACATCCAAGGCTGTGGGAAGGTGTACGGCGATCGCAGCAACCTGACA CGTCACTTGCGCTGGCATACCGGGGAGAAGCCGCATATTTGCCACATCCAAGGCT GTGGGAAGGTGTACGGCAAGAACTGGAAGCTCCAGGCGCACTTGCGCTGGCATA 44
CCGGTGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTC GTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAG AAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAA GTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCA ATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTA TAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGT CGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGT CTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAG GAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGC TGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACA TTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTC CAGCTTACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGA GGCGGTTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAG GCGGTGGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTG GCTCAGGCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAAC TGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAAT ATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAAT GAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGT GGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACG GTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCA AGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATA TCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTT TTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTA AATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTG GTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAAT TTAATAACGGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAG GAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCG CATAG
Sample ID
pBM2-SBE7-3
ZFs
52-116-75-10-86-139-2-17
Target DNA
GAA GAC GCT ATA GGC AAC GAA AAG
45
Table.12. Nucleotide Sequence of pBM2-SBE7-4 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGAAATTTGCCTGCCCTGAGTGTCCTAAGCG CTTCATGCGTAGCGACAACCTGACACAGCATATCAAGACCCACACCGGGGAGAA GCCTTTTCAATGCAACCAGTGCGGTGCGAGCTTCACGCAAAAGGGCAACTTACTG CGCCACATCAAGCTGCATACCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGA AAGAGTTTTAGCGATTCTGGAAATCTCAGAGTGCACCAGAGAACACATACCGGG GAGAAGCCGCATATTTGCCACATCCAAGGCTGTGGGAAGGTGTACGGCGATCGT AGCCACCTGACACGTCACTTGCGCTGGCATACCGGGGAGAAGCCTTACAAATGC CCAGAATGTGGAAAGAGTTTTAGCCAAAAGTCTTCCCTCATCGCTCACCAGAGA ACACATACCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGC ACTTCTGGAGAACTCGTGAGACACCAGAGAACACATACCGGGGAGCGGCCATTT ATGTGTACCTGGTCATACTGTGGGAAACGCTTCACAGATCGCAGCAACCTGACAC GTCACAAACGTACACACACCGGGGAGAAGCCGCATATTTGCCACATCCAAGGCT GTGGGAAGGTGTACGGCCAACGCAGCAACCTGGTACGTCACTTGCGCTGGCATA CCGGTGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTC GTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAG AAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAA GTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCA ATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTA TAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGT CGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGT CTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAG GAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGC TGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACA TTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTC CAGCTTACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGA GGCGGTTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAG GCGGTGGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTG GCTCAGGCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAAC TGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAAT ATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAAT GAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGT GGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACG GTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCA AGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATA TCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTT TTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTA AATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTG GTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAAT TTAATAACGGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAG 46
GAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCG CATAG
Sample ID
pBM2-SBE7-4
ZFs
52-116-75-61-86-72-19-1
Target DNA
GAA GAC GCT ATA GGC AAC GAA AAG
Table.13. Nucleotide Sequence of pBM2-SBE9-1 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGAAAGAG TTTTAGCGATCCTGGACATCTTGTGAGACACCAGAGAACACATACCGGGGAGAA GCCTTACATGTGCTCCGAGTGCGGGCGGGGTTTCAGCCAGAAGAGCAACCTCAT CATCCACCAGCGCACCCACACCGGGGAGAAGCCTTACAAGTGCGAGGAGTGCGG CAAGGCGTTCAACCAGAGCAGCACGTTGACGCGGCACAAGATCGTGCACACCGG GGAGAAGCCTTACACCTGCAAGCAGTGCGGCAAGGCATTCAGCGTGTCCTCCTC CCTGAGGCGGCACGAGACCACCCACACCGGGGAGAAGCCTTACAAATGCCCAGA ATGTGGAAAGAGTTTTAGCAGGAATGATGCACTCACAGAACACCAGAGAACACA TACCGGGGAGCGGCCATTTATGTGTACCTGGTCATACTGTGGGAAACGCTTCACA GATCGTAGCCACCTCGCACGTCACAAACGTACACACACCGGGGAGAAGCCTTAC AAATGCCCAGAATGTGGAAAGAGTTTTAGCTCTCCTGCTGATCTCACAAGACACC AGAGAACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGCGGCAAGACGT TCAGCGTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACCGGTGGATCCCA ACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAA ATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAG GATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATA GAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCG GATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTA TAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCA AACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCT GTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAG CTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGT AGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGA GGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCAGCTTACTGC ATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGGCGGTTCTGG TGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGCGGTGGTGGT 47
TCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCTCAGGCGGT GGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGGAGGAGAAG AAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAA TTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGG AATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAA ACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTG GATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAA ATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAAT GAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGA GTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCAC TAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATG ATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGC GAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGAAGTCTGCTA ACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Sample ID
pBM2-SBE9-1
ZFs
61-121-127-138-102-26-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
Table.14. Nucleotide Sequence of pBM2-SBE9-2 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGAAAGAG TTTTAGCAGGTCTGATGAACTCGTGAGACACCAGAGAACACATACCGGGGAGAA GCCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCCAATCTGGAAATCTCAC AGAACACCAGAGAACACATACCGGGGAGCGGCCATTTATGTGTACCTGGTCATA CTGTGGGAAACGCTTCACACAAAGCGGTGCCCTTGCTCGTCACAAACGTACACA CACCGGGGAGAAGCCTTACACCTGCAAGCAGTGCGGCAAGGCATTCAGCGTGTC CTCCTCCCTGAGGCGGCACGAGACCACCCACACCGGGGAGAAGAAATTTGCCTG CCCTGAGTGTCCTAAGCGCTTCATGCGTAGCGATGCACTGCGTGAGCATATCAAG ACCCACACCGGGGAGCGGCCATTTATGTGTACCTGGTCATACTGTGGGAAACGCT TCACAGATCGTAGCCACCTCGCACGTCACAAACGTACACACACCGGGGAGAAGC CTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCTCTCCTGCTGATCTCACAAG ACACCAGAGAACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGCGGCAA GACGTTCAGCGTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACCGGTGGA TCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAA 48
TTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCA CTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGG ATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATAC TGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGA GGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAA AATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCA TCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTA CAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTT AGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACC TTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCAGCTT ACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGGCGGT TCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGCGGTG GTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCTCAG GCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGGAGG AGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGA ATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTA ATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAA GGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGAT CGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGA TGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCC TAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTG TGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATAT CACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAA ATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAAC GGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGAAGTCT GCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Sample ID
pBM2-SBE9-2
ZFs
66-89-29-138-55-26-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
49
Table.15. Nucleotide Sequence of pBM2-SBE9-3 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGCGGCCATTTATGTGTACCTGGTCATACTGTGGG AAACGCTTCACAGATCGTAGCCACCTCGCACGTCACAAACGTACACACACCGGG GAGAAGCCTTACATGTGCTCCGAGTGCGGGCGGGGTTTCAGCCAGAAGAGCAAC CTCATCATCCACCAGCGCACCCACACCGGGGAGCGGCCATTTATGTGTACCTGGT CATACTGTGGGAAACGCTTCACACAAAGCGGTGCCCTTGCTCGTCACAAACGTAC ACACACCGGGGAGAAGCCTTACACCTGCAAGCAGTGCGGCAAGGCATTCAGCGT GTCCTCCTCCCTGAGGCGGCACGAGACCACCCACACCGGGGAGAAGCCTTACAA ATGCCCAGAATGTGGAAAGAGTTTTAGCAGGAATGATGCACTCACAGAACACCA GAGAACACATACCGGGGAGCGGCCATTTATGTGTACCTGGTCATACTGTGGGAA ACGCTTCACAGATCGTAGCCACCTCGCACGTCACAAACGTACACACACCGGGGA GAAGCCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCTCTCCTGCTGATCTC ACAAGACACCAGAGAACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGC GGCAAGACGTTCAGCGTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACC GGTGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGT CATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAA ATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGT TTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAAT TTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATA GCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCG AAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCT ATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGA AACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTG TTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATT AACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCA GCTTACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGG CGGTTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGC GGTGGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCT CAGGCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGG AGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATAT TGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAG GTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGAT CAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGT GATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGC AGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCA ACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTT ATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAAT CATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTG GAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTA ATAACGGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGA 50
AGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCA TAG
Sample ID
pBM2-SBE9-3
ZFs
26-121-29-138-102-26-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
Table.16. Nucleotide Sequence of pBM2-SBE9-4 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGCCGCATATTTGCCACATCCAAGGCTGTGG GAAGGTGTACGGCGATCGTAGCCACCTGACACGTCACTTGCGCTGGCATACCGG GGAGAAGCCTTACGTGTGCAGCAAGTGCGGCAAGGCTTTCACCCAGAGCAGCAA CCTCACCGTGCACCAGAAGATCCACACCGGGGAGAAGCCTTACAAGTGCGAGGA GTGCGGCAAGGCGTTCAACCAGAGCAGCACGTTGACGCGGCACAAGATCGTGCA CACCGGGGAGAAGCCGCATATTTGCCACATCCAAGGCTGTGGGAAGGTGTACGG CCGTAGCGATGCACTGACACGTCACTTGCGCTGGCATACCGGGGAGAAGAAATT TGCCTGCCCTGAGTGTCCTAAGCGCTTCATGCGTAGCGATGCACTGCGTGAGCAT ATCAAGACCCACACCGGGGAGCGGCCATTTATGTGTACCTGGTCATACTGTGGG AAACGCTTCACAGATCGTAGCCACCTCGCACGTCACAAACGTACACACACCGGG GAGAAGCCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCTCTCCTGCTGATC TCACAAGACACCAGAGAACACATACCGGGGAGAAGCCTTACGAGTGCAACTACT GCGGCAAGACGTTCAGCGTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACA CCGGTGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTC GTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAG AAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAA GTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCA ATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTA TAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGT CGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGT CTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAG GAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGC TGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACA TTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTC 51
CAGCTTACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGA GGCGGTTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAG GCGGTGGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTG GCTCAGGCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAAC TGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAAT ATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAAT GAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGT GGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACG GTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCA AGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATA TCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTT TTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTA AATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTG GTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAAT TTAATAACGGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAG GAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCG CATAG
Sample ID
pBM2-SBE9-4
ZFs
10-125-127-15-55-26-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
Table.17. Nucleotide Sequence of pBM2-SBE9-5 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGAAAGAG TTTTAGCGATCCTGGACATCTTGTGAGACACCAGAGAACACATACCGGGGAGAA GCCTTACATGTGCTCCGAGTGCGGGCGGGGTTTCAGCCAGAAGAGCAACCTCAT CATCCACCAGCGCACCCACACCGGGGAGAAGCCTTACAAGTGCGAGGAGTGCGG CAAGGCGTTCAACCAGAGCAGCACGTTGACGCGGCACAAGATCGTGCACACCGG GGAGAAGCCGCATATTTGCCACATCCAAGGCTGTGGGAAGGTGTACGGCCGTAG CGATGCACTGACACGTCACTTGCGCTGGCATACCGGGGAGAAGCCTTACAAATG CCCAGAATGTGGAAAGAGTTTTAGCAGGAATGATGCACTCACAGAACACCAGAG AACACATACCGGGGAGCGGCCATTTATGTGTACCTGGTCATACTGTGGGAAACG CTTCACAGATCGTAGCCACCTCGCACGTCACAAACGTACACACACCGGGGAGAA GCCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCTCTCCTGCTGATCTCACA 52
AGACACCAGAGAACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGCGGC AAGACGTTCAGCGTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACCGGTG GATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATA AATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTC CACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTAT GGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTAT ACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCG GAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAG AAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATC CATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAAC TACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTC TTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAAC CTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCAGCT TACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGGCGG TTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGCGGT GGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCTCA GGCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGGAG GAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTG AATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGT AATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCA AGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGA TCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAG ATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACC CTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTT GTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCAT ATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAG AAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATA ACGGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGAAGT CTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Sample ID
pBM2-SBE9-5
ZFs
61-121-127-15-102-26-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
53
Table.18. Nucleotide Sequence of pBM2-SBE9-6 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGCCGCATATTTGCCACATCCAAGGCTGTGG GAAGGTGTACGGCGATCGTAGCCACCTGACACGTCACTTGCGCTGGCATACCGG GGAGAAGCCTTACATGTGCTCCGAGTGCGGGCGGGGTTTCAGCCAGAAGAGCAA CCTCATCATCCACCAGCGCACCCACACCGGGGAGAAGCCTTACAAGTGCGAGGA GTGCGGCAAGGCGTTCAACCAGAGCAGCACGTTGACGCGGCACAAGATCGTGCA CACCGGGGAGCGGCCATTTATGTGTACCTGGTCATACTGTGGGAAACGCTTCACA CGTAGCGATGCACTGAGCCGTCACAAACGTACACACACCGGGGAGAAGCCTTAC AAATGCCCAGAATGTGGAAAGAGTTTTAGCAGGAATGATGCACTCACAGAACAC CAGAGAACACATACCGGGGAGAAGAAATTTGCCTGCCCTGAGTGTCCTAAGCGC TTCATGGATCGTAGCCACCTCGCACGTCATATCAAGACCCACACCGGGGAGAAG CCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCTCTCCTGCTGATCTCACAA GACACCAGAGAACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGCGGCA AGACGTTCAGCGTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACCGGTGG ATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAA ATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCC ACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATG GATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATA CTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGG AGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGA AAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCC ATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACT ACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCT TAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAAC CTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCAGCT TACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGGCGG TTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGCGGT GGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCTCA GGCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGGAG GAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTG AATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGT AATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCA AGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGA TCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAG ATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACC CTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTT GTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCAT ATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAG AAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATA ACGGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGAAGT 54
CTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Sample ID
pBM2-SBE9-6
ZFs
10-121-127-31-102-45-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
Table.19. Nucleotide Sequence of pBM2-SBE9-7 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGAAAGAG TTTTAGCGATCCTGGACATCTTGTGAGACACCAGAGAACACATACCGGGGAGAA GCCTTACGTGTGCAGCAAGTGCGGCAAGGCTTTCACCCAGAGCAGCAACCTCAC CGTGCACCAGAAGATCCACACCGGGGAGAAGCCTTACAAGTGCGAGGAGTGCGG CAAGGCGTTCAACCAGAGCAGCACGTTGACGCGGCACAAGATCGTGCACACCGG GGAGAAGCCTTACACCTGCAAGCAGTGCGGCAAGGCATTCAGCGTGTCCTCCTC CCTGAGGCGGCACGAGACCACCCACACCGGGGAGAAGCCTTACAAATGCCCAGA ATGTGGAAAGAGTTTTAGCAGGAATGATGCACTCACAGAACACCAGAGAACACA TACCGGGGAGAAGAAATTTGCCTGCCCTGAGTGTCCTAAGCGCTTCATGGATCGT AGCCACCTCGCACGTCATATCAAGACCCACACCGGGGAGAAGCCTTACAAATGC CCAGAATGTGGAAAGAGTTTTAGCTCTCCTGCTGATCTCACAAGACACCAGAGA ACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGCGGCAAGACGTTCAGC GTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACCGGTGGATCCCAACTAG TCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATG TGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAG AATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGT AAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCT CCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATC TGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACAC GAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAAC GGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAG CTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAG AGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAG TGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCAGCTTACTGCATCGCC CGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGGCGGTTCTGGTGGTGG CGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGCGGTGGTGGTTCTGGC GGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCTCAGGCGGTGGTGGT 55
GTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCT GAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAA TTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTT TATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGA CGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACT AAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAA CGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGG TGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCA CTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGT AATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAG CCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAA ACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGAAGTCTGCTAACATGCG GTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Sample ID
pBM2-SBE9-7
ZFs
61-125-127-138-102-45-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
Table.20. Nucleotide Sequence of pBM2-SBE9-8 ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGA CGATAAATCTAGACCCGGGGAGAAGCCGCATATTTGCCACATCCAAGGCTGTGG GAAGGTGTACGGCGATCGTAGCCACCTGACACGTCACTTGCGCTGGCATACCGG GGAGAAGCCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCCAATCTGGAAA TCTCACAGAACACCAGAGAACACATACCGGGGAGAAGCCTTACAAGTGCGAGGA GTGCGGCAAGGCGTTCAACCAGAGCAGCACGTTGACGCGGCACAAGATCGTGCA CACCGGGGAGAAGCCTTACACCTGCAAGCAGTGCGGCAAGGCATTCAGCGTGTC CTCCTCCCTGAGGCGGCACGAGACCACCCACACCGGGGAGAAGAAATTTGCCTG CCCTGAGTGTCCTAAGCGCTTCATGCGTAGCGATGCACTGCGTGAGCATATCAAG ACCCACACCGGGGAGCGGCCATTTATGTGTACCTGGTCATACTGTGGGAAACGCT TCACAGATCGTAGCCACCTCGCACGTCACAAACGTACACACACCGGGGAGAAGC CTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCTCTCCTGCTGATCTCACAAG ACACCAGAGAACACATACCGGGGAGAAGCCTTACGAGTGCAACTACTGCGGCAA GACGTTCAGCGTGAGCTCCACGCTCATTCGCCACCAGCGCATCCACACCGGTGGA TCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAA TTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCA 56
CTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGG ATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATAC TGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGA GGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAA AATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCA TCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTA CAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTT AGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACC TTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTTGTCCAGCTT ACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTCAGGTGGAGGCGGT TCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGGTGGTTCAGGCGGTG GTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGTGGCGGTGGCTCAG GCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAAAGTGAACTGGAGG AGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGA ATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTA ATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAA GGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGAT CGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGA TGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCC TAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTG TGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATAT CACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAA ATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAAC GGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGGGCAGAGGAAGTCT GCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCGCCGGCGCATAG
Sample ID
pBM2-SBE9-8
ZFs
10-89-127-138-55-26-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
Table.20A. Nucleotide Sequence of pTWINI-8ZFs-FokI-D ATGTACAAGGATGACGACGATAAATCTAGACCCGGGGAGAAGCCTTACAAATGC CCAGAATGTGGAAAGAGTTTTAGCGATCCTGGACATCTTGTGAGACACCAGAGA ACACATACCGGGGAGAAGCCTTACATGTGCTCCGAGTGCGGGCGGGGTTTCAGC CAGAAGAGCAACCTCATCATCCACCAGCGCACCCACACCGGGGAGAAGCCTTAC AAGTGCGAGGAGTGCGGCAAGGCGTTCAACCAGAGCAGCACGTTGACGCGGCAC 57
AAGATCGTGCACACCGGGGAGAAGCCTTACACCTGCAAGCAGTGCGGCAAGGCA TTCAGCGTGTCCTCCTCCCTGAGGCGGCACGAGACCACCCACACCGGGGAGAAG CCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCAGGAATGATGCACTCACA GAACACCAGAGAACACATACCGGGGAGCGGCCATTTATGTGTACCTGGTCATAC TGTGGGAAACGCTTCACAGATCGTAGCCACCTCGCACGTCACAAACGTACACAC ACCGGGGAGAAGCCTTACAAATGCCCAGAATGTGGAAAGAGTTTTAGCTCTCCT GCTGATCTCACAAGACACCAGAGAACACATACCGGGGAGAAGCCTTACGAGTGC AACTACTGCGGCAAGACGTTCAGCGTGAGCTCCACGCTCATTCGCCACCAGCGC ATCCACACCGGTGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCT GAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAA TTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTT TATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGA CGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACT AAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAA CGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGG TGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCA CTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGT AATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAG CCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAA ACTTTGTCCAGCTTACTGCATCGCCCGGGGGCGGCGGTTCTGGTGGCGGTGGTTC AGGTGGAGGCGGTTCTGGTGGTGGCGGTTCAGGTGGTGGTGGTTCAGGTGGCGG TGGTTCAGGCGGTGGTGGTTCTGGCGGAGGCGGTTCAGGTGGCGGTGGTTCTGGT GGCGGTGGCTCAGGCGGTGGTGGTGTATTGCGATCGGTCGTACAACTAGTCAAA AGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCT CATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTC TTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACA TTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATT GATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAA TTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACA AACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATT TAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACA CGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTT TAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGAC GGAAATTTAATAACGGCGAGATAAACTTTGGAGGTGGCAGCGGTGGCGGTGAGG GCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCATTCG CCGGCGTGCAGCCGGTGTATAGCCTTCGTGTCGACACGGCAGACCACGCGTTTAT CACGAACGGGTTCGTCAGCCACGCTACTGGCCTCACCGGTCTGAACTCAGGCCTC ACGACAAATCCTGGTGTATCCGCTTGGCAGGTCAACACAGCTTATACTGCGGGAC AATTGGTCACATATAACGGCAAGACGTATAAATGTTTGCAGCCCCACACCTCCTT GGCAGGATGGGAACCATCCAACGTTCCTGCCTTGTGGCAGCTTCAATGA
58
Sample ID
pTWINI-8ZFs-FokI-D
ZFs
61-121-127-138-102-26-78-139
Target DNA
GCT ACA GGC CTG GTG GTA CAA GGC
Table.21. Protein Sequence of pBM1 Vector Met T T I N L D S K L G S Q L V K S E L E E K K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E NQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQL T R L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop Sample ID
pBM1-FokI
Table.22. Protein Sequence of pc2XB-Linker SRPGVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGG S G G G G S G G G G S G G G G S G G G G S G G G G V L R S V V T G G Sc
Sample ID
pc3XB-Linker
Table.23. Protein Sequence of pBM2 Vector Met T T I N L D S K L G S Q L V K S E L E E K K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E 59
NQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQL T R L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N NGEINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEE K K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G AStop
Sample ID
pBM2-FokI-D
Table.24. Protein Sequence of pBM2-SBE7-1 Met G P K K K R K V A A A D Y K D D D D K S R P G E K K F A C P E C P K R F Met R S D N L T Q H I K T H T G E K P F Q C N Q C G A S F T Q K G N L L R H I K L HTGEKPYKCPECGKSFSDSGNLRVHQRTHTGEKKFACPECP K R F Met D R S H L A R H I K T H T G E K P Y K C P E C G K S F S Q K S S L I A H Q RTHTGEKPYECNYCGKTFSVSSTLIRHQRIHTGEKPYKCKQC GKAFGCPSNLRRHGRTHTGEKPHICHIQGCGKVYGQRSNLV RHLRWHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIA R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q TRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTR L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G EINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEK K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
60
Table.25. Protein Sequence of pBM2-SBE7-2 Met G P K K K R K V A A A D Y K D D D D K S R P G E K K F A C P E C P K R F Met R S D N L T Q H I K T H T G E K P F Q C N Q C G A S F T Q K G N L L R H I K L HTGEKPYKCPECGKSFSDSGNLRVHQRTHTGEKPHICHIQGC GKVYGDRSHLTRHLRWHTGEKPYKCPECGKSFSQKSSLIAH QRTHTGEKPYECNYCGKTFSVSSTLIRHQRIHTGEKPYKCKQ CGKAFGCPSNLRRHGRTHTGEKPHICHIQGCGKVYGKNWKL QAHLRWHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEI A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q TRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTR L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G EINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEK K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
Table.26. Protein Sequence of pBM2-SBE7-3
Met G P K K K R K V A A A D Y K D D D D K S R P G E K K F A C P E C P K R F Met R S D N L T Q H I K T H T G E K P F Q C N Q C G A S F T Q K G N L L R H I K L HTGEKPYKCPECGKSFSDSGNLRVHQRTHTGEKPHICHIQGC GKVYGDRSHLTRHLRWHTGEKPYKCPECGKSFSQKSSLIAH QRTHTGEKPYECNYCGKTFSVSSTLIRHQRIHTGEKPHICHI QGCGKVYGDRSNLTRHLRWHTGEKPHICHIQGCGKVYGKN WKLQAHLRWHTGGSQLVKSELEEKKSELRHKLKYVPHEYIE L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V EENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKA Q L T R L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K 61
FNNGEINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSE L E E K K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S SVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELL I G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
Table.27. Protein Sequence of pBM2-SBE7-4 Met G P K K K R K V A A A D Y K D D D D K S R P G E K K F A C P E C P K R F Met R S D N L T Q H I K T H T G E K P F Q C N Q C G A S F T Q K G N L L R H I K L HTGEKPYKCPECGKSFSDSGNLRVHQRTHTGEKPHICHIQGC GKVYGDRSHLTRHLRWHTGEKPYKCPECGKSFSQKSSLIAH Q R T H T G E K P Y K C P E C G K S F S T S G E L V R H Q R T H T G E R P F Met C T WSYCGKRFTDRSNLTRHKRTHTGEKPHICHIQGCGKVYG QR SNLVRHLRWHTGGSQLVKSELEEKKSELRHKLKYVPHEYIE L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E MetQ R Y V E ENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQ L T R L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F NNGEINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSEL E E K K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
62
Table.28. Protein Sequence of pBM2-SBE9-1 Met G P K K K R K V A A A D Y K D D D D K S R P G E K P Y K C P E C G K S F S D P G H L V R H Q R T H T G E K P Y Met C S E C G R G F S Q K S N L I I H Q R T H T G EKPYKCEECGKAFNQSSTLTRHKIVHTGEKPYTCKQCGKAF SVSSSLRRHETTHTGEKPYKCPECGKSFSRNDALTEHQRTHT G E R P F Met C T W S Y C G K R F T D R S H L A R H K R T H T G E K P Y K C P E C GKSFSSPADLTRHQRTHTGEKPYECNYCGKTFSVSSTLIRHQ RIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNST Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K HINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHI T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G E I N F VQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEKKSEL R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y GYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTK AYSGGYNL P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S V T E F K F L F V S G H F K G N Y K A Q L T R L N H I T N C N G A V L S V E E L L I G G E Met I KAGTLTLEEVRRKFNNGEINFGGGSGGGEGRGSLLTCGDVE E N P G P F A G A Stop
Table.29. Protein Sequence of pBM2-SBE9-2 Met G P K K K R K V A A A D Y K D D D D K S R P G E K P Y K C P E C G K S F S R SDELVRHQRTHTGEKPYKCPECGKSFSQSGNLTEHQRTHTG E R P F Met C T W S Y C G K R F T Q S G A L A R H K R T H T G E K P Y T C K Q C G K A F S V S S S L R R H E T T H T G E K K F A C P E C P K R F Met R S D A L R E H I K T H T G E R P F Met C T W S Y C G K R F T D R S H L A R H K R T H T G E K P Y K CPECGKSFSSPADLTRHQRTHTGEKPYECNYCGKTFSVSSTL IRHQRIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIA R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E MetQ R Y V E E N Q T RNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRL N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G E INFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEKK S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K 63
VYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGY N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S V T E F KFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGG E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
Table.30. Protein Sequence of pBM2-SBE9-3 Met G P K K K R K V A A A D Y K D D D D K S R P G E R P F Met C T W S Y C G K R F T D R S H L A R H K R T H T G E K P Y Met C S E C G R G F S Q K S N L I I H Q R T H T G E R P F Met C T W S Y C G K R F T Q S G A L A R H K R T H T G E K P Y T C K QCGKAFSVSSSLRRHETTHTGEKPYKCPECGKSFSRNDALTE H Q R T H T G E R P F Met C T W S Y C G K R F T D R S H L A R H K R T H T G E K P YKCPECGKSFSSPADLTRHQRTHTGEKPYECNYCGKTFSVSS TLIRHQRIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIE I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N QTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLT R L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N GEINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEE K K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F FMet K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
64
Table.31. Protein Sequence of pBM2-SBE9-4 Met G P K K K R K V A A A D Y K D D D D K S R P G E K P H I C H I Q G C G K V Y GDRSHLTRHLRWHTGEKPYVCSKCGKAFTQSSNLTVHQKIH TGEKPYKCEECGKAFNQSSTLTRHKIVHTGEKPHICHIQGCG K V Y G R S D A L T R H L R W H T G E K K F A C P E C P K R F Met R S D A L R E H I K T H T G E R P F Met C T W S Y C G K R F T D R S H L A R H K R T H T G E K P Y KCPECGKSFSSPADLTRHQRTHTGEKPYECNYCGKTFSVSST LIRHQRIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEI A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q TRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTR L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G EINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEK K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
Table.32. Protein Sequence of pBM2-SBE9-5 Met G P K K K R K V A A A D Y K D D D D K S R P G E K P Y K C P E C G K S F S D P G H L V R H Q R T H T G E K P Y Met C S E C G R G F S Q K S N L I I H Q R T H T G EKPYKCEECGKAFNQSSTLTRHKIVHTGEKPHICHIQGCGKV YGRSDALTRHLRWHTGEKPYKCPECGKSFSRNDALTEHQRT H T G E R P F Met C T W S Y C G K R F T D R S H L A R H K R T H T G E K P Y K C P ECGKSFSSPADLTRHQRTHTGEKPYECNYCGKTFSVSSTLIR HQRIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIAR N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T RNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRL N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G E INFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEKK S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K 65
VYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGY N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S V T E F KFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGG E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
Table.33. Protein Sequence of pBM2-SBE9-6 Met G P K K K R K V A A A D Y K D D D D K S R P G E K P H I C H I Q G C G K V Y G D R S H L T R H L R W H T G E K P Y Met C S E C G R G F S Q K S N L I I H Q R T H T G E K P Y K C E E C G K A F N Q S S T L T R H K I V H T G E R P F Met C T W S YCGKRFTRSDALSRHKRTHTGEKPYKCPECGKSFSRNDALT E H Q R T H T G E K K F A C P E C P K R F Met D R S H L A R H I K T H T G E K P Y KCPECGKSFSSPADLTRHQRTHTGEKPYECNYCGKTFSVSST LIRHQRIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEI A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q TRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTR L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G EINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEK K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T C G D V E E N P G P F A G A Stop
Table.34. Protein Sequence of pBM2-SBE9-7 Met G P K K K R K V A A A D Y K D D D D K S R P G E K P Y K C P E C G K S F S D PGHLVRHQRTHTGEKPYVCSKCGKAFTQSSNLTVHQKIHTG EKPYKCEECGKAFNQSSTLTRHKIVHTGEKPYTCKQCGKAF 66
SVSSSLRRHETTHTGEKPYKCPECGKSFSRNDALTEHQRTHT G E K K F A C P E C P K R F Met D R S H L A R H I K T H T G E K P Y K C P E C G K SFSSPADLTRHQRTHTGEKPYECNYCGKTFSVSSTLIRHQRI HTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQ D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H INPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHIT N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G E I N F V QLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEKKSELR H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G YRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPI G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S V T E F K F L F V S G H F K G N Y K A Q L T R L N H I T N C N G A V L S V E E L L I G G E Met I K AGTLTLEEVRRKFNNGEINFGGGSGGGEGRGSLLTCGDVEE N P G P F A G A Stop
Table.35. Protein Sequence of pBM2-SBE9-8 Met G P K K K R K V A A A D Y K D D D D K S R P G E K P H I C H I Q G C G K V Y GDRSHLTRHLRWHTGEKPYKCPECGKSFSQSGNLTEHQRTH TGEKPYKCEECGKAFNQSSTLTRHKIVHTGEKPYTCKQCGK A F S V S S S L R R H E T T H T G E K K F A C P E C P K R F Met R S D A L R E H I K T H T G E R P F Met C T W S Y C G K R F T D R S H L A R H K R T H T G E K P Y K C PECGKSFSSPADLTRHQRTHTGEKPYECNYCGKTFSVSSTLI RHQRIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIA R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q TRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTR L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N N G EINFVQLTASPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGVLRSVVQLVKSELEEK K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S S VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI G G E Met I K A G T L T L E E V R R K F N N G E I N F G G G S G G G E G R G S L L T 67
C G D V E E N P G P F A G A Stop
Table.35A. Protein Sequence of pTWINI-FokI-D Met Y K D D D D K S R P G E K P Y K C P E C G K S F S D P G H L V R H Q R T H T G E K P Y Met C S E C G R G F S Q K S N L I I H Q R T H T G E K P Y K C E E C G K A F NQSSTLTRHKIVHTGEKPYTCKQCGKAFSVSSSLRRHETTHT G E K P Y K C P E C G K S F S R N D A L T E H Q R T H T G E R P F Met C T W S Y C GKRFTDRSHLARHKRTHTGEKPYKCPECGKSFSSPADLTRH QRTHTGEKPYECNYCGKTFSVSSTLIRHQRIHTGGSQLVKSE L E E K K S E L R H K L K Y V P H E Y I E L I E I A R N S T Q D R I L EMet K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E N Q T R N K H I N P N E W W K V Y P S SVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELL I G G E Met I K A G T L T L E E V R R K F N N G E I N F V Q L T A S P G G G G S G G GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGVLRSVVQLVKSELEEKKSELRHKLKYVPHEYIELI E I A R N S T Q D R I L E Met K V Met E F F Met K V Y G Y R G K H L G G S R K P D G A I Y T V G S P I D Y G V I V D T K A Y S G G Y N L P I G Q A D E Met Q R Y V E E NQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQL T R L N H I T N C N G A V L S V E E L L I G G E Met I K A G T L T L E E V R R K F N NGEINFGGGSGGGEGRGSLLTCGDVEENPGPFAGVQPVYSL RVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNT AYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQL Q Stop
68
References 1. Miller J,; McLachlan AD,; Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985,4(6):1609–1614. 2. Pavletich NP,; Pabo CO. Zinc Finger-DNA Recognition: Crystal Structure of a Zif268-DNA Complex at 2.1A° Science. 1991,252:809–817. 3. Pabo CO,; Peisach E,; Grant RA. Design and selection of novel Cys2His2 zinc finger proteins. Annu. Rev. Biochem. 2001,70:313–340. 4. Shukla, V. K.; Doyon, Y.; Miller, J. C.; Dekelver, R. C.; Moehle, E. A.; Worden, S. E.; Mitchell, J. C.; Arnold, N. L. et al.Precise genome modification in the crop species Zea mays using zincfinger nucleases. Nature 2009,459 (7245): 437–41. 5. Stephen C. Ekker.Zinc Finger-based knock out punches for zebrafish genes. Zebrafish. June 2008, 5(2): 121-123 6. Carroll, D. Progress and prospects: Zinc-finger nucleases as gene therapy agents. Gene Ther. 2008,15 (22), 1463-1468. 7. Geurts, A. M.; Cost, G. J.; Freyvert, Y.; Zeitler, B.; Miller, J. C.; Choi, V. M.; Jenkins, S. S.; Wood, A. et al. Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases. Science 2009,325 (5939): 433–433. 8. Tebas, Pablo et al. (February 2009). Autologous T-Cells Genetically Modified at the CCR5 Gene by Zinc Finger Nucleases SB-728 for HIV (Zinc-Finger) 9. Pâques F,; Haber JE. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999,63(2):349–404 10. West SC,; Chappell C,; Hanakahi LA,; Masson JY,; Mcllwraith MJ,; Van Dyck E. Double-strand break repair in human cells. Cold spring Harb Symp Quant Biol. 2000,65: 315-321 11. McCormack, W.T., L.W. Tjoelker & C.B. Thompson. Avian B cell development: Generation of an immunoglobulin repertoire by gene conversion. Annu. Rev. Imm.1991, 9:219-241. 12. Segal DJ,; Dreier B,; Beerli RR,; Barbas C.F. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. Proc. Natl Acad. Sci. USA. 1999,96:2758–2763. 13. Bitinaite, J.; Aggarwal, A. K.; Schildkraut, I. FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci USA 95 (18): 10570–5. 14. Cathomen T, Joung JK (July 2008). "Zinc-finger nucleases: the next generation emerges". Mol. Ther. 1998,16 (7): 1200–7. 15. Cornu TI,; Thibodeau-Beganny S,; Guhl E et al. DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Molecular Therapy. 2008, 16(2): 352–358. 16. J.C. Miller,; M.C. Holmes,; J. Wang,; D.Y. Guschin,; Y.-L. Lee,; L. Rupniewski,; C.M. Beausejour,; A.J. Waite,; N.S. Wang,; K.A. Kim,; P.D. Gregory,; C.O. Pabo and E.J. Rebar. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol., 2007, 25 pp. 778–785. 17. Szczepek, M.,; Brondani, V.,; Bü chel, J.; Serrano, L.,; Segal, D.J., & Cathomen, T. Structurebased redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases Nat Biotechnol 2007, 25: 786-793. 18. Tomoaki Mori,; Ikuko Kagatsume,; Kazuki Shinomiya,; Yasuhiro Aoyama,; Takashi Sera. Sandwiched Zinc-finger nucleases harboring a single-chain FokI dimer as a Dna-cleavage domain. Biochemical and Biophysical Research Communications, 2009, Volume 390, Issue 3, Pages 694-697 19. Michal Minczuk,; Monika A. Papworth,; Jeffrey C. Miller,; Michael P. Murphy,; Aaron Klug. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA. Nucleic Acids Res. 2008, 36(12): 3926–3938. 20. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature. 2011, 475,217–221.
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21. Wood A. J. et al. Targeted genome editing across species using ZFNs and TALENs. Science. 2011, 333, 307. 22. Eva-Maria Händel,; Stephen Alwin,; Toni Cathomen. Expanding or Restricting the Target Site Repertoire of Zinc-finger Nuclease: The inter-domain Linker as a Major Determinant of Target Site Selectivity. Mol Ther. 2009, 17(1): 104–111.
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Glossary Assembly PCR Synthesizing longer fragments of DNA using more than one same or different DNA templates with the help of specially designed flanking primers. The method used the polymerase chain reaction directed by more than one template DNA and primers flanked with two consecutive DNA templates in a defined order.
Colony PCR For the identification of successfully transformed E.coli cells the mixture of transformed and untransformed cells were spread on the plate containing Agar media with the speciefic antibiotics. The E.coli cells with the Plasmid having gene of interest and specific resistance will survive on the Agar media and develop into colonies. These colonies were later picked , grown and later used as a template for the PCR Reaction for the evaluation of presence of gene of interest. Such PCR Reaction is termed as Colony PCR and it verify the presence of gene of interest into a plasmid residing into a specific strain of bacteria.
Gel Purification The term Gel purification is used for the purification of DNA samples from the Agarose. It involves excision of DNA from the Agarose Gel, Dissolving, Binding to a DNA binding Column, Washing and Elution.
Ligation Ligation is used to join the restricted DNA fragments usually to make a circular Plasmid DNA or to synthesize a longer DNA fragment. 71
PCR Purification The term PCR purification means the removal of reaction mixture components other than DNA such as enzymes and salts. The technique usually applies after the Restriction reaction in order to purify the DNA from Restriction enzymes and Buffer components.
PCR Reactions Polymerase chain reactions were carried out in order to amplify the target DNA region using a pair of short oligo nucleotides (primers) from a small amount of template DNA. The DNA synthesis reaction is catalyzed by Taq-Polymerase (Solgent) in case using a bacterial colony as a template and pfu-Polymerase (Solgent) in case of using synthetic DNA or purified Plasmids.
Restriction The term usually refers the cleavage of Synthetic or Natural DNA molecule from specific positions using corresponding recognizing Endonucleases or Restriction enzymes. Restriction produce products of DNA with specific sticky or non-specific blunt ends.
Sequencing of Data During the study the sequencing were done using standard sequencing methods at Macrogen. The genes of interest usually incorporated into a vector and later multiplied using basic molecular techniques , purified and sent for sequencing with one or two primers depending on the size of sequencing read.
Transformation The Incorporation of certain gnene into Plasmid vectors is a handy way to investigate and preserve the gene. The usual product of cloning is a vector with desired gene as well as a gene 72
with resistant marker. The Chemically competent bacterial cells (e.g, E.coli C2566 NEB) can take up plasmids from a mixture by Heat Shock. These plasmids can transform Chemically competent Bacterial cells (E.coli C2566 NEB) to develop resistance against a particular antibiotics provided through the plasmid and hence the gene of interest can survive and multiply into a transformed E.coli for generations to come.
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Targeted Genome Editing using Single Chain Zinc Finger Dimeric Nuclease Muhammad Mustafa, Hwangbeom Kim and Duhee Bang* Synthetic Biology Laboratory, Department of Chemistry, Yonsei University
Introduction An engineered zinc finger nucleases (ZFNs) have fusions between the DNA cleavage domain of FokI and a custom-designed DNA binding domain. Each Zinc Finger can Recognize three nucleotides sequence of DNA. Two Zinc Finger Arrays with FokI catalytic domain can bind to a specific site with an appropriate spacer to make a Zinc Finger Nuclease. The Zinc Finger Nucleases or ZFN are one of the most handsome tools in business to effectively create a Double Stranded DNA break (DSB). A DSB can be manipulate to generate a frame shift gene knockout by NHEJ or to incorporate exogenous DNA fragment by Homologous Recombination (HR). Much of the work in this field is carried out to evolve new designs and synthetic strategies to reduce the time and cost of production of ZFN.
The problems associated with Assembly of multi ZFs using typical cloning methods takes days to finish with 3 or 4 ZF encoding genes assembled for functional use. PCR Based Methods are not usually work well to assemble the components with similar repeating units as ZF encoding proteins have and things are too messy when we need to increase the ZF number up to 8 ZF. So the combination of Hierarchical assembly and cloning would work to end with multi ZF array up to 8Zfs within 6 days. PCR Based synthesis of 4ZFs array was optimized and done in 3 days which were used in turn to make 8ZFs array .
Synthesis of Multi-ZFs Assembly
3A 1A
Fig 3: (3A) PCR based ZiF assembly (3B) Cloning scheme. .
Linker
1B
TARGET DNA
Fig 1: (1A) Typical Zinc finger Nuclease functional Diamer with 12bp recognition site. (1B) Proposed enhanced single Chain Zinc Finger Dimeric Nuclease with 24bp Recognition
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pc3XB ZF1
Synthesis of Single Chain FokI- Nuclease
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pBM1 Linker
ZF5 HindIII
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AgeI & HindIII Restriction enzyme digestion
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XmaI & HindIII Restriction enzyme digestion
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First PCR reaction FokI
First PCR reaction
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Second PCR reaction F1
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ZF1 ZF2 ZF3 ZF4 ZF5 ZF6 ZF7 ZF8
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1. XbaI & BamHI Restriction enzyme digestion 2. Ligation
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Third PCR reaction AscI
NheI FokI
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pBM2 ZF1 ZF2 ZF3 ZF4 ZF5 ZF6 ZF7 ZF8
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2C 1820bp
Fig 4: (4A) Cloning scheme of 8ZFs with FokI dimer (4B) Electrophoresis showing Cloning of 8ZFs in pBM2 Vector
4B
1362bp 694bp
1000bp 830bp 200bp
Conclusion Fig 2: Scheme and Results for the Synthesis of single chain FokI-Diameric Nuclease. (A) Vectors and primers for the first PCR Reaction. Flanking Regions with same color. (B) Second PCR Reaction with Primers to assemble two FokI together with a flexible 10 units of GGGGS amino acids. (C) Gel Electrophoresis of PCR Products
http://chem.yonsei.ac.kr/~duhee
Single Chain Zinc finger Nuclease have 650 bp repeating unit was synthesize using PCR based Method. The flexible amino acid linker can be of varied length to allow optimized dimerization to form functional nuclease. Up to 8ZFs can be assembled linearly to synthesize a 24bp recognition site using PCR based method supported by cloning method in 6 days
SyntheticBiologyLab.
