Zinc finger nuclease

Zinc-finger nucleases (ZFNs) are artificial restrictionenzymes generated by fusing a zinc finger DNA-bindingdomain to a DNA-cleavage domain. Zinc finger domainscan be engineered to target desired DNA sequences andthis enables zinc-finger nucleases to target unique se-quences within complex genomes. By taking advantageof endogenous DNA repair machinery, these reagents canbe used to precisely alter the genomes of higher organ-isms.

1 DNA-cleavage domain

A pair of two ZFNs with three zinc fingers each are shown in-troducing a double-strand break. Subsequent to this, the doublestrand break is being repaired through either homologous recom-bination or non-homologous end joining.[1]

The non-specific cleavage domain from the type IIsrestriction endonuclease FokI is typically used as thecleavage domain in ZFNs.[2] This cleavage domain mustdimerize in order to cleave DNA[3] and thus a pairof ZFNs are required to target non-palindromic DNAsites. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. In order to allow thetwo cleavage domains to dimerize and cleave DNA, thetwo individual ZFNs must bind opposite strands of DNAwith their C-termini a certain distance apart. The mostcommonly used linker sequences between the zinc fingerdomain and the cleavage domain requires the 5' edge ofeach binding site to be separated by 5 to 7 bp.[4]

Several different protein engineering techniques havebeen employed to improve both the activity and speci-ficity of the nuclease domain used in ZFNs. Directedevolution has been employed to generate a FokI variant

with enhanced cleavage activity that the authors dubbed“Sharkey”.[5] Structure-based design has also been em-ployed to improve the cleavage specificity of FokI bymodifying the dimerization interface so that only the in-tended heterodimeric species are active.[6][7][8][9]

2 DNA-binding domain

The DNA-binding domains of individual ZFNs typicallycontain between three and six individual zinc finger re-peats and can each recognize between 9 and 18 basepairs.If the zinc finger domains are perfectly specific for theirintended target site then even a pair of 3-finger ZFNs thatrecognize a total of 18 basepairs can, in theory, target asingle locus in a mammalian genome.Various strategies have been developed to engineerCys2His2 zinc fingers to bind desired sequences.[10]These include both “modular assembly” and selectionstrategies that employ either phage display or cellular se-lection systems.The most straightforward method to generate new zinc-finger arrays is to combine smaller zinc-finger “modules”of known specificity. The most common modular assem-bly process involves combining three separate zinc fingersthat can each recognize a 3 basepair DNA sequence togenerate a 3-finger array that can recognize a 9 basepairtarget site. Other procedures can utilize either 1-finger or2-finger modules to generate zinc-finger arrays with sixor more individual zinc fingers. The main drawback withthis procedure is the specificities of individual zinc fin-gers can overlap and can depend on the context of thesurrounding zinc fingers and DNA. Without methods toaccount for this “context dependence”, the standard mod-ular assembly procedure often fails unless it is used torecognize sequences of the form (GNN)N.[11]

Numerous selection methods have been used to generatezinc-finger arrays capable of targeting desired sequences.Initial selection efforts utilized phage display to selectproteins that bound a given DNA target from a largepool of partially randomized zinc-finger arrays. More re-cent efforts have utilized yeast one-hybrid systems, bacte-rial one-hybrid and two-hybrid systems, and mammaliancells. A promising new method to select novel zinc-fingerarrays utilizes a bacterial two-hybrid system and has beendubbed “OPEN” by its creators.[12] This system combinespre-selected pools of individual zinc fingers that wereeach selected to bind a given triplet and then utilizes a sec-

1

2 3 APPLICATIONS

ond round of selection to obtain 3-finger arrays capableof binding a desired 9-bp sequence. This system was de-veloped by the Zinc-Finger Consortium as an alternativeto commercial sources of engineered zinc-finger arrays.(see: Zinc finger chimera for more info on zinc fingerselection techniques)

3 Applications

Zinc finger nucleases have become useful reagents formanipulating the genomes of many plants and animalsincluding arabidopsis,[13][14] tobacco,[15][16] soybean,[17]corn,[18] Drosophila melanogaster,[19] C. elegans,[20] seaurchin,[21] silkworm,[22] zebrafish,[23] frogs,[24] mice,[25]rats,[26] rabbits,[27] pigs,[28] cattle,[29] and various typesof mammalian cells.[30] Zinc finger nucleases have alsobeen used in a mouse model of haemophilia[31] and anongoing clinical trial is evaluating Zinc finger nucleasesthat disrupt the CCR5 gene in CD4+ human T-cells asa potential treatment for HIV/AIDS. ZFNs are also usedfor the creation of a new generation of genetic diseasemodels called isogenic human disease models.

3.1 Disabling an allele

ZFNs can be used to disable dominant mutations in het-erozygous individuals by producing double-strand breaks(DSBs) in the DNA (see Genetic recombination) in themutant allele, which will, in the absence of a homologoustemplate, be repaired by non-homologous end-joining(NHEJ). NHEJ repairs DSBs by joining the two ends to-gether and usually produces no mutations, provided thatthe cut is clean and uncomplicated. In some instances,however, the repair will be imperfect, resulting in deletionor insertion of base-pairs, producing frame-shift and pre-venting the production of the harmful protein.[32] Mul-tiple pairs of ZFNs can also be used to completely re-move entire large segments of genomic sequence.[33] Tomonitor the editing activity, a PCR of the target areawill amplify both alleles and, if one contains an inser-tion, deletion, or mutation, it will result in a heterduplexsingle-strand bubble that cleavage assays can easily de-tect. ZFNs have also been used tomodify disease-causingalleles in triplet repeat disorders. Expanded CAG/CTGrepeat tracts are the genetic basis for more than a dozeninherited neurological disorders including Huntington’sdisease, myotonic dystrophy, and several spinocerebel-lar ataxias. It has been demonstrated in human cells thatZFNs can direct double-strand breaks (DSBs) to CAG re-peats and shrink the repeat from long pathological lengthsto short, less toxic lengths.[34]

Recently, a group of researchers have successfully ap-plied the ZFN technology to genetically modify the golpigment gene and the ntl gene in zebrafish embryo. Spe-cific zinc-finger motifs were engineered to recognize dis-

tinct DNA sequences. The ZFN-encoding mRNA wasinjected into one-cell embryos and a high percentage ofanimals carried the desired mutations and phenotypes.Their research work demonstrated that ZFNs can specif-ically and efficiently create heritable mutant alleles at lociof interest in the germ line, and ZFN-induced alleles canbe propagated in subsequent generations.Similar research of using ZFNs to create specific muta-tions in zebrafish embryo has also been carried out byother research groups. The kdr gene in zebra fish en-codes for the vascular endothelial growth factor-2 recep-tor. Mutagenic lesions at this target site was induced us-ing ZFN technique by a group of researchers in US. Theysuggested that the ZFN technique allows straightforwardgeneration of a targeted allelic series of mutants; it doesnot rely on the existence of species-specific embryonicstem cell lines and is applicable to other vertebrates, espe-cially those whose embryos are easily available; finally, itis also feasible to achieve targeted knock-ins in zebrafish,therefore it is possible to create human disease modelsthat are heretofore inaccessible.

3.2 Allele editing

ZFNs are also used to rewrite the sequence of an allele byinvoking the homologous recombination (HR) machin-ery to repair the DSB using the supplied DNA fragmentas a template. The HR machinery searches for homol-ogy between the damaged chromosome and the extra-chromosomal fragment and copies the sequence of thefragment between the two broken ends of the chromo-some, regardless of whether the fragment contains theoriginal sequence. If the subject is homozygous for thetarget allele, the efficiency of the technique is reducedsince the undamaged copy of the allele may be used as atemplate for repair instead of the supplied fragment.

3.3 Gene therapy

The success of gene therapy depends on the effi-cient insertion of therapeutic genes at the appropriatechromosomal target sites within the human genome,without causing cell injury, oncogenic mutations or animmune response. The construction of plasmid vectorsis simple and straightforward. Custom-designed ZFNsthat combine the non-specific cleavage domain (N) ofFokI endonuclease with zinc-finger proteins (ZFPs) of-fer a general way to deliver a site-specific DSB to thegenome, and stimulate local homologous recombinationby several orders of magnitude. This makes targeted genecorrection or genome editing a viable option in humancells. Since ZFN-encoding plasmids could be used totransiently express ZFNs to target a DSB to a specificgene locus in human cells, they offer an excellent wayfor targeted delivery of the therapeutic genes to a pre-selected chromosomal site. The ZFN-encoding plasmid-

3

based approach has the potential to circumvent all theproblems associated with the viral delivery of therapeu-tic genes.[35] The first therapeutic applications of ZFNsare likely to involve ex vivo therapy using a patients ownstem cells. After editing the stem cell genome, the cellscould be expanded in culture and reinserted into the pa-tient to produce differentiated cells with corrected func-tions. The initial targets will likely include the causes ofmonogenic diseases such as the IL2Rγ gene and the b-globin gene for gene correction and CCR5 gene for mu-tagenesis and disablement.[32]

4 Potential Problems

4.1 Off-target Cleavage

If the zinc finger domains are not specific enough for theirtarget site or they do not target a unique site within thegenome of interest, off-target cleavage may occur. Suchoff-target cleavage may lead to the production of enoughdouble-strand breaks to overwhelm the repair machin-ery and, as a consequence, yield chromosomal rearrange-ments and/or cell death. Off-target cleavage events mayalso promote random integration of donor DNA.[32] Twoseparate methods have been demonstrated to decreaseoff-target cleavage for 3-finger ZFNs that target two ad-jacent 9-basepair sites.[36] Other groups use ZFNs with4, 5 or 6 zinc fingers that target longer and presumablyrarer sites and such ZFNs could theoretically yield lessoff-target activity. A comparison of a pair of 3-fingerZFNs and a pair of 4-finger ZFNs detected off-targetcleavage in human cells at 31 loci for the 3-finger ZFNsand at 9 loci for the 4-finger ZFNs.[37] Whole genome se-quencing of C. elegans modified with a pair of 5-fingerZFNs found only the intended modification and a dele-tion at a site “unrelated to the ZFN site” indicating thispair of ZFNs was capable of targeting a unique site in theC. elegans genome.[20]

4.2 Immunogenicity

For more details on this topic, see Adaptive immuneresponse.

As with many foreign proteins inserted into the humanbody, there is a risk of an immunological response againstthe therapeutic agent and the cells in which it is active.Since the protein will need to be expressed only tran-siently, however, the time over which a response may de-velop is short.[32] Liu et al respectively target ZFNick-ases to the endogenous b-casein(CSN2) locus stimu-lates lysostaphin and human lysozyme gene addition byhomology-directed repair and derive secrete lysostaphincows.[38] [39]

5 Prospects

The ability to precisely manipulate the genomes of plants,animals and insects has numerous applications in basic re-search, agriculture, and human therapeutics. Using ZFNsto modify endogenous genes has traditionally been a dif-ficult task due mainly to the challenge of generating zincfinger domains that target the desired sequence with suf-ficient specificity. Improved methods of engineering zincfinger domains and the availability of ZFNs from a com-mercial supplier now put this technology in the hands ofincreasing numbers of researchers. Several groups arealso developing other types of engineered nucleases in-cluding engineered homing endonucleases [40] [41] and nu-cleases based on engineered TAL effectors. [42] [43] TALeffector nucleases (TALENs) are particularly interestingbecause TAL effectors appear to be very simple to engi-neer [44] [45] and TALENs can be used to target endoge-nous loci in human cells.[46] But to date no one has re-ported the isolation of clonal cell lines or transgenic or-ganisms using such reagents. One type of ZFN, knownas SB-728-T, has been tested for potential application inthe treatment of HIV.[47]

6 Zinc-finger Nickases

Zinc-finger nickases (ZFNickases) are created by inac-tivating the catalytic activity of one ZFN monomer inthe ZFN dimer required for double-strand cleavage.[48]ZFNickases demonstrate strand-specific nicking activityin vitro and thus provide for highly specific single-strandbreaks in DNA.[48] These SSBs undergo the same cellularmechanisms for DNA that ZFNs exploit, but they show asignificantly reduced frequency of mutagenic NHEJ re-pairs at their target nicking site. This reduction providesa bias for HR-mediated gene modifications. ZFNickasescan induce targeted HR in cultured human and livestockcells, although at lower levels than corresponding ZFNsfrom which they were derived because nicks can be re-paired without genetic alteration.[49] [38]A major limita-tion of ZFN-mediated gene modifications is the competi-tion between NHEJ and HR repair pathways. Regardlessof the presence of a DNA donor construct, both repairmechanisms can be activated following DSBs induced byZFNs. Thus, ZFNickases is the first plausible attempt atengineering a method to favor the HR method of DNArepair as opposed to the error-prone NHEJ repair. By re-ducing NHEJ repairs, ZFNickases can thereby reduce thespectrum of unwanted off-target alterations. The ease bywhich ZFNickases can be derive from ZFNs provides agreat platform for further studies regarding the optimiza-tion of ZFNickases and possibly increasing their levelsof targeted HR while still maintain their reduced NHEJfrequency.

4 7 ZINC FINGER NUCLEASE TREATMENT OF HIV

7 Zinc Finger Nuclease Treatmentof HIV

A. Targeting and Editing Host Cellular Co-Receptors forHIVSince antiretroviral therapy requires a lifelong treatmentregimen, research to find more permanent cures for HIVinfection is currently underway.[50] It is possible to syn-thesize zinc finger nucleotides with zinc finger compo-nents that selectively (almost selectively) bind to specificportions of DNA. It has also been observed that 20% ofthe Caucasian population possess what is referred to asthe CCR5-Δ32 mutation (frequency of 0.0808 for ho-mozygous allele) that prevents the CCR5 protein, whichis the main means of viral access into the cell, from beingexpressed on the surface of their T-cells.[51][52][53][54][55]Individuals who are homozygous for this mutation are im-mune to HIV strains that utilize the CCR5 receptor in or-der to gain access to the cell while those who are heterozy-gous for this mutation have been found to have reducedplasma viral load in addition to a delayed progression toAIDS.[56] By combining these facts, researchers have pro-posed a novel method of treatment for HIV. This methodattempts to treat the infection by introducing the CCR5-Δ32 mutation and, as a consequence, resulting in the ex-pression of nonfunctional CCR5 co-receptors on CD4+ Tcells, providing immunity against infection.[1][56][57]

The zinc finger nucleases that have been synthesizedfor this treatment are manufactured by combining FokIType II restriction endonucleases with engineered zincfingers.[1][58] The number of zinc fingers attached to theendonuclease controls the specificity of the ZFN sincethey are engineered to preferentially bind to specific basesequences in DNA. Each ZFN ismade up ofmultiple zincfingers and one nuclease enzyme.[1]

B. Targeting Proviral HIV DNAA recent and unique application of ZFN-technology totreat HIV has emerged whose focus is to target notthe host genome, but rather proviral HIV DNA, formutagenesis.[59] The authors of this work have drawntheir inspiration from the innate defense mechanismagainst bacteria-infecting-viruses called bacteriophages,present amongst those bacteria endowed with restric-tion modification (R-M) systems. These bacteria se-cret a restriction enzyme (REase) that recognizes andrepetitively cleaves around palindromic sequences withinthe xenogenic DNAs of the bacteriophages or simplyphages, until the same is disabled. Further support forthis approach resides in the fact that, the human genomecomprises in large part remnants of retroviral genomesthat have been inactivated by several mechanisms, someof whose action resembles that of ZFN. It should notbe surprising, therefore, that the initial work leading tothe application of ZFN technology in this manner re-volved around and involved the isolation and testing ofHIV/SIV targeting bacteria-derived REases, whose non-

specificity (due to their short recognition sequences) un-fortunately, rendered them toxic to the host genome. Thelatter-potential host-genome toxicity posed by the rawbacteria-derived REases limited their application to ex-vivo modalities for HIV prevention, namely synthetic orlive microbicides. Subsequently, however, the uniquespecificity offered by ZFNs was quickly recognized andharnessed, paving way for a novel strategy for attack-ing HIV in-vivo (through target mutagenesis of proviralHIV DNA) that is similar to the manner by which bac-teria equipped with R-M systems do, to disable the for-eign DNAs of in-coming phage-genomes. Because la-tent proviral HIV DNA resident in resting memory CD4cells forms the major barrier to the eradication of HIVby highly active antiviral therapy (HAART), it is specu-lated that this approach may offer a 'functional cure” forHIV. Both ex-vivo (manipulation of stem or autologousT cell precursors) and in-vivo delivery platforms are be-ing explored. It is also hoped that, when applied to non-HIV infected persons, this strategy could offer a genomicvaccine against HIV and other viruses. Similar work isongoing for high-risk HPVs (with the intent of revers-ing cervical neoplasia) [60] as well as with HSV-2 (withthe goal of achieving a complete cure for genital herpes)[61][62][63][64][65][66][67][68][69][70][71]

7.1 Zinc Finger Binding

The exact constitution of the ZFNs that are to be usedto treat HIV is still unknown. The binding of ZFNs forthe alteration of the Zif268 genelink, however, has beenwell-studied and is outlined below in order to illustratethe mechanism by which the zinc finger domain of ZFNsbind to DNA.[72][73]

The amino terminus of the alpha helix portion of zincfingers targets the major grooves of the DNA helix andbinds near the CCR5 gene positioning FokI in a suitablelocation for DNA cleavage.[1][72][73]

Zinc fingers are repeated structural protein motifs withDNA recognition function that fit in the major groovesof DNA.[72] Three zinc fingers are positioned in a semi-circular or C-shaped arrangement.[73] Each zinc fingeris made up of anti-parallel beta sheets and an alphahelix, held together by a zinc ion and hydrophobicresidues.[72][73]

The zinc atom is constrained in a tetrahedral conforma-tion through the coordination of Cys3, Cys6, His19, andHis23 and Zinc – Sulfur bond distance of 2.30 +/- 0.05Angstroms and Zinc – Nitrogen bond distances of 2.0 +/-0.05 Angstroms.[73][74][75]

Each zinc finger has an arginine (arg) amino acid pro-truding from the alpha helix, which forms a hydrogenbond with Nitrogen 7 and Oxygen 6 of the guanine (gua)that is located at the 3’ end of the binding site.[72][73][75]The arg-gua bond is stabilized by Aspartic acid from a2nd residue, which positions the long chain of arginine

7.4 Limitations 5

through a hydrogen bond salt bridge interaction.[72][76]

In residue 3 of the 2nd (i.e., middle) zinc finger,histidine49 forms a hydrogen bond with a co-planarguanine in base pair 6. The stacking of Histidine againstThymine in base pair 5 limits the conformational abil-ity of Histidine49 leading to increased specificity for thehistidine-guanine hydrogen bond.[72][73]

At the 6th residue, fingers 1 and 3 have arginine donatinga pair of charged hydrogen bonds to Nitrogen 7 and Oxy-gen 6 of guanine at the 5’ end enhancing the site recogni-tion sequence of zinc fingers.[72][73]

Contacts with DNA backboneThe Histidine coordinated to the zinc atom, which is alsothe seventh residue in the alpha helix of the zinc fingers,coordinates the Zinc ion through its Nε and hydrogenbonds with phosphodiester oxygen through Nδ on the pri-mary DNA strand.[72][73][76]

In addition to histidine, a conserved arginine on the sec-ond beta strand of the zinc fingers makes contact with thephosphodiester oxygen on the DNA strand.[72][73][76]

Also Serine 75 on the third finger hydrogen bonds to thephosphate between base pairs 7 and 8, as the only back-bone contact with the secondary strand of DNA.[72][73][76]

7.2 Nuclease Dimerization and Cleavage

It has been discovered that FokI has no intrinsic speci-ficity in its cleavage of DNA and that the zinc fin-ger recognition domain confers selectivity to zinc fingernucleases.[1][58]

Specificity is provided by dimerization, which decreasesthe probability of off-site cleavage. Each set of zinc fin-gers is specific to a nucleotide sequence on either sideof the targeted gene 5-7 bp separation between nucleasecomponents.[1]

The dimerization of two ZFNs is required to produce thenecessary double-strand break within the CCR5 gene be-cause the interaction between the FokI enzyme and DNAis weak.[57] This break is repaired by the natural repairmechanisms of the cell, specifically non-homologous endjoining.[57]

7.3 Introducing the CCR5 Mutation

Introducing genome alterations depends upon either ofthe two natural repair mechanisms of a cell: non-homologous end joining (NHEJ) and homology-directedrepair (HDR).[57] Repair through NHEJ comes about bythe ligation of the end of the broken strands and, uponthe occurrence of an error, can produce small insertionsand deletions. HDR, on the other hand, makes use of ahomologous DNA strand in order to repair and gene andmaking use of this repair mechanism and providing the

desired nucleotide sequence allows for gene insertion ormodification.[57]

The main DSB repair pathway in mammals (that occursin the absence of a homologous nucleotide base sequencethat can be used by a homologous recombination mecha-nism is through non-homologous end joining (NHEJ).[77]NHEJ, although capable of restoring a damaged gene, iserror-prone.[77] DSB are, therefore, introduced into thegene until an error in its repair occurs at which pointZFNs are no longer able to bind and dimerize and themutation is complete.[77] In order to accelerate this pro-cess, exonucleases can be introduced to digest the ends ofthe strands generated at DSBs.[77]

7.4 Limitations

Increasing the number of zinc fingers increases the speci-ficity by increasing the number of base pairs that the ZFNcan bind to.[1] However too many zinc fingers can lead tooff-target binding and thus offsite cleavage.[1] This is dueto an increased likelihood of zinc fingers binding to partsof the genome outside of the gene of interest.Current ZFN treatments focus on the CCR5 gene as noknown side effects result from altering CCR5.[78] Thereare strains of HIV that are able to use CXCR4 to enter thehost cell, bypassing CCR5 altogether.[78] The same geneediting technology has been applied to CXCR4 alone andin combination with CCR5 [79][80]

Several issues exist with this experimental treatment. Oneissue lies in ensuring that the desired repair mechanismis the one that is used to repair the DSB following geneaddition.[81] Another issue with the disruption of theCCR5 gene is that CXCR4-specific or dual-tropic strainsare still able to access the cell.[81] This method can pre-vent the progression of HIV infection.To employ the ZFNs in clinical settings the following cri-teria need to be met: i) High specificity of DNA-binding– Correlates with better performance and less toxicityof ZFNs. Engineered ZFNs take into account positionaland context-dependent effects of zinc fingers to increasespecificity.[82] ii) Enable allosteric activation of FokI oncebound to DNA in order for it to produce only the requiredDSB.[82] iii) In order to deliver two different zinc fingernuclease subunits and donor DNA to the cell, the vectorsthat are used need to be improved to decrease the riskof mutagenesis.[82] These include adeno-associated virusvectors, integrase-deficient lentiviral vectors and aden-ovirus type 5 vectors.[82] iv) Transient expression of ZFNswould be preferred over permanent expression of theseproteins in order to avoid ‘off-target’ effects.[82] v) Duringgene targeting, genotoxicity associated with high expres-sion of ZFNs might lead to cell apoptosis and thus needsto be thoroughly verified in vitro and in vivo transforma-tion assays.[82]

6 9 REFERENCES

7.5 Administration of Treatment

The cells in which the mutations are induced ex vivo arefiltered out from lymphocytes by apheresis to produceanalogous lentiviral engineered CD4+ T-cells.[83] Theseare re-infused into the body as a single dose of 1 X 1010gene modified analogous CD4+ T-cells.[83] A viral vectoris used to deliver the ZFNs that will induce the desiredmutation into the cells. Conditions that promote this pro-cess are carefully monitored ensuring the production ofCCR5 strain HIV-resistant T cells.[84]

The Berlin PatientTimothy Ray Brown, who underwent a bone mar-row transplant in 2007 to treat leukemia, had HIVsimultaneously.[85] Soon after the operation the HIVdropped to undetectable levels.[85] This is a result of thebone marrow donor being homozygous for the CCR5-Δ32 mutation.[85] This new mutation conferred a resis-tance to HIV in the recipient, eventually leading to analmost complete disappearance of HIV particles in hisbody.[85] After nearly 2 years without antiretroviral drugtherapy, HIV could still not be detected in any of histissues.[85][86] Though this method has been effective atreducing the level of infection, the risks associated withbone marrow transplants outweighs its potential value asa treatment for HIV.[52]

8 See also

• Genome editing with engineered nucleases

• Zinc finger

• Gene targeting

• Zinc finger protein

• Zinc finger chimera

• Protein engineering

• Genome engineering

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10 11 EXTERNAL LINKS

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10 Further reading• Mandell JG, Barbas CF (July 2006). “Zinc Fin-ger Tools: custom DNA-binding domains fortranscription factors and nucleases”. NucleicAcids Res. 34 (Web Server issue): W516–23.doi:10.1093/nar/gkl209. PMC 1538883. PMID16845061.

• Porteus MH, Carroll D (August 2005). “Gene tar-geting using zinc finger nucleases”. Nat. Biotech-nol. 23 (8): 967–73. doi:10.1038/nbt1125. PMID16082368.

• Doyon Y, McCammon JM, Miller JC et al.(June 2008). “Heritable Targeted Gene Disrup-tion in Zebrafish Using Designed Zinc Finger Nu-cleases”. Nat. Biotechnol. 26 (6): 702–8.doi:10.1038/nbt1409. PMC 2674762. PMID18500334.

• Meng X, Noyes MB, Zhu LJ, Lawson ND, WolfeSA (June 2008). “Targeted gene inactivation in ze-brafish using engineered zinc finger nucleases”. Nat.Biotechnol. 26 (6): 695–701. doi:10.1038/nbt1398.PMC 2502069. PMID 18500337.

11 External links• Zinc finger selector

• Zinc Finger Consortium website

• Zinc Finger Consortium materials from Addgene

• A commercial supplier of ZFNs

11

12 Text and image sources, contributors, and licenses

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