World Library  
Flag as Inappropriate
Email this Article

Zinc finger nuclease

Article Id: WHEBN0006476735
Reproduction Date:

Title: Zinc finger nuclease  
Author: World Heritage Encyclopedia
Language: English
Subject: Isogenic human disease models, CRISPR, Genome editing, Knockout rat, Recombinant AAV mediated genome engineering
Collection: Engineered Proteins, Zinc Proteins
Publisher: World Heritage Encyclopedia

Zinc finger nuclease

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.


  • Domains 1
    • DNA-binding domain 1.1
    • DNA-cleavage domain 1.2
  • Applications 2
    • Disabling an allele 2.1
    • Allele editing 2.2
    • Gene therapy 2.3
  • Potential problems 3
    • Off-target cleavage 3.1
    • Immunogenicity 3.2
  • Prospects 4
  • Zinc-finger nickases 5
  • Zinc finger nuclease treatment of HIV 6
    • Host cellular co-receptors for HIV 6.1
    • Proviral HIV DNA 6.2
    • Zinc finger binding 6.3
    • Nuclease dimerization and cleavage 6.4
    • Introducing the CCR5 mutation 6.5
    • Limitations 6.6
    • Administration of treatment 6.7
  • See also 7
  • References 8
  • Further reading 9
  • External links 10


A zinc finger nuclease is a site-specific endonuclease designed to bind and cleave DNA at specific positions. There are two protein domains. The first domain is the DNA binding domain, which consists of eukaryotic transcription factors and contain the zinc finger. The second domain is the nuclease domain, which consists of the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA.

DNA-binding domain

The DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 basepairs. If the zinc finger domains are perfectly specific for their intended target site then even a pair of 3-finger ZFNs that recognize a total of 18 basepairs can, in theory, target a single locus in a mammalian genome.

Various strategies have been developed to engineer Cys2His2 zinc fingers to bind desired sequences.[1] These include both "modular assembly" and selection strategies that employ either phage display or cellular selection 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 assembly process involves combining three separate zinc fingers that can each recognize a 3 basepair DNA sequence to generate a 3-finger array that can recognize a 9 basepair target site. Other procedures can utilize either 1-finger or 2-finger modules to generate zinc-finger arrays with six or more individual zinc fingers. The main drawback with this procedure is the specificities of individual zinc fingers can overlap and can depend on the context of the surrounding zinc fingers and DNA. Without methods to account for this "context dependence", the standard modular assembly procedure often fails unless it is used to recognize sequences of the form (GNN)N.[2]

Numerous selection methods have been used to generate zinc-finger arrays capable of targeting desired sequences. Initial selection efforts utilized phage display to select proteins that bound a given DNA target from a large pool of partially randomized zinc-finger arrays. More recent efforts have utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. A promising new method to select novel zinc-finger arrays utilizes a bacterial two-hybrid system and has been dubbed "OPEN" by its creators.[3] This system combines pre-selected pools of individual zinc fingers that were each selected to bind a given triplet and then utilizes a second round of selection to obtain 3-finger arrays capable of binding a desired 9-bp sequence. This system was developed by the Zinc-Finger Consortium as an alternative to commercial sources of engineered zinc-finger arrays.

(see: Zinc finger chimera for more info on zinc finger selection techniques)

DNA-cleavage domain

A pair of ZFNs, each with three zinc fingers binding to target DNA, are shown introducing a double-strand break, at the FokI domain, depicted in yellow. Subsequently, the double strand break is shown as being repaired through either homology-directed repair or non-homologous end joining.[4]

The non-specific cleavage domain from the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFNs.[5] This cleavage domain must dimerize in order to cleave DNA[6] and thus a pair of ZFNs are required to target non-palindromic DNA sites. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. In order to allow the two cleavage domains to dimerize and cleave DNA, the two individual ZFNs must bind opposite strands of DNA with their C-termini a certain distance apart. The most commonly used linker sequences between the zinc finger domain and the cleavage domain requires the 5' edge of each binding site to be separated by 5 to 7 bp.[7]

Several different protein engineering techniques have been employed to improve both the activity and specificity of the nuclease domain used in ZFNs. Directed evolution has been employed to generate a FokI variant with enhanced cleavage activity that the authors dubbed "Sharkey".[8] Structure-based design has also been employed to improve the cleavage specificity of FokI by modifying the dimerization interface so that only the intended heterodimeric species are active.[9][10][11][12]


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

Disabling an allele

ZFNs can be used to disable dominant mutations in heterozygous individuals by producing double-strand breaks (DSBs) in the DNA (see Genetic recombination) in the mutant allele, which will, in the absence of a homologous template, be repaired by non-homologous end-joining (NHEJ). NHEJ repairs DSBs by joining the two ends together and usually produces no mutations, provided that the cut is clean and uncomplicated. In some instances, however, the repair will be imperfect, resulting in deletion or insertion of base-pairs, producing frame-shift and preventing the production of the harmful protein.[32] Multiple pairs of ZFNs can also be used to completely remove entire large segments of genomic sequence.[33] To monitor the editing activity, a PCR of the target area will amplify both alleles and, if one contains an insertion, deletion, or mutation, it will result in a heterduplex single-strand bubble that cleavage assays can easily detect. ZFNs have also been used to modify disease-causing alleles in triplet repeat disorders. Expanded CAG/CTG repeat tracts are the genetic basis for more than a dozen inherited neurological disorders including Huntington’s disease, myotonic dystrophy, and several spinocerebellar ataxias. It has been demonstrated in human cells that ZFNs can direct double-strand breaks (DSBs) to CAG repeats and shrink the repeat from long pathological lengths to short, less toxic lengths.[34]

Recently, a group of researchers have successfully applied the ZFN technology to genetically modify the gol pigment gene and the ntl gene in zebrafish embryo. Specific zinc-finger motifs were engineered to recognize distinct DNA sequences. The ZFN-encoding mRNA was injected into one-cell embryos and a high percentage of animals carried the desired mutations and phenotypes. Their research work demonstrated that ZFNs can specifically and efficiently create heritable mutant alleles at loci of interest in the germ line, and ZFN-induced alleles can be propagated in subsequent generations.

Similar research of using ZFNs to create specific mutations in zebrafish embryo has also been carried out by other research groups. The kdr gene in zebra fish encodes for the vascular endothelial growth factor-2 receptor. Mutagenic lesions at this target site was induced using ZFN technique by a group of researchers in US. They suggested that the ZFN technique allows straightforward generation of a targeted allelic series of mutants; it does not rely on the existence of species-specific embryonic stem cell lines and is applicable to other vertebrates, especially those whose embryos are easily available; finally, it is also feasible to achieve targeted knock-ins in zebrafish, therefore it is possible to create human disease models that are heretofore inaccessible.

Allele editing

ZFNs are also used to rewrite the sequence of an allele by invoking the homologous recombination (HR) machinery to repair the DSB using the supplied DNA fragment as a template. The HR machinery searches for homology between the damaged chromosome and the extra-chromosomal fragment and copies the sequence of the fragment between the two broken ends of the chromosome, regardless of whether the fragment contains the original sequence. If the subject is homozygous for the target allele, the efficiency of the technique is reduced since the undamaged copy of the allele may be used as a template for repair instead of the supplied fragment.

Gene therapy

The success of gene therapy depends on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, oncogenic mutations or an immune response. The construction of plasmid vectors is simple and straightforward. Custom-designed ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc-finger proteins (ZFPs) offer a general way to deliver a site-specific DSB to the genome, and stimulate local homologous recombination by several orders of magnitude. This makes targeted gene correction or genome editing a viable option in human cells. Since ZFN-encoding plasmids could be used to transiently express ZFNs to target a DSB to a specific gene locus in human cells, they offer an excellent way for targeted delivery of the therapeutic genes to a pre-selected chromosomal site. The ZFN-encoding plasmid-based approach has the potential to circumvent all the problems associated with the viral delivery of therapeutic genes.[35] The first therapeutic applications of ZFNs are likely to involve ex vivo therapy using a patients own stem cells. After editing the stem cell genome, the cells could be expanded in culture and reinserted into the patient to produce differentiated cells with corrected functions. The initial targets will likely include the causes of monogenic diseases such as the IL2Rγ gene and the b-globin gene for gene correction and CCR5 gene for mutagenesis and disablement.[32]

Potential problems

Off-target cleavage

If the zinc finger domains are not specific enough for their target site or they do not target a unique site within the genome of interest, off-target cleavage may occur. Such off-target cleavage may lead to the production of enough double-strand breaks to overwhelm the repair machinery and, as a consequence, yield chromosomal rearrangements and/or cell death. Off-target cleavage events may also promote random integration of donor DNA.[32] Two separate methods have been demonstrated to decrease off-target cleavage for 3-finger ZFNs that target two adjacent 9-basepair sites.[36] Other groups use ZFNs with 4, 5 or 6 zinc fingers that target longer and presumably rarer sites and such ZFNs could theoretically yield less off-target activity. A comparison of a pair of 3-finger ZFNs and a pair of 4-finger ZFNs detected off-target cleavage in human cells at 31 loci for the 3-finger ZFNs and at 9 loci for the 4-finger ZFNs.[37] Whole genome sequencing of C. elegans modified with a pair of 5-finger ZFNs found only the intended modification and a deletion at a site "unrelated to the ZFN site" indicating this pair of ZFNs was capable of targeting a unique site in the C. elegans genome.[20]


As with many foreign proteins inserted into the human body, there is a risk of an immunological response against the therapeutic agent and the cells in which it is active. Since the protein will need to be expressed only transiently, however, the time over which a response may develop is short.[32] Liu et al. respectively target ZFNickases to the endogenous b-casein(CSN2) locus stimulates lysostaphin and human lysozyme gene addition by homology-directed repair and derive secrete lysostaphin cows.[38][39]


The ability to precisely manipulate the genomes of plants, animals and insects has numerous applications in basic research, agriculture, and human therapeutics. Using ZFNs to modify endogenous genes has traditionally been a difficult task due mainly to the challenge of generating

  • Zinc finger selector
  • Zinc Finger Consortium website
  • Zinc Finger Consortium materials from Addgene
  • A commercial supplier of ZFNs

External links

Further reading

  1. ^
  2. ^
  3. ^
  4. ^ a b c d e f g h i Carroll, D. Genome engineering with zinc-finger nucleases. Genetics Society of America, 2011, 188(4), pp 773-782.
  5. ^
  6. ^
  7. ^
  8. ^
  9. ^
  10. ^
  11. ^
  12. ^
  13. ^
  14. ^
  15. ^
  16. ^
  17. ^
  18. ^
  19. ^
  20. ^ a b
  21. ^
  22. ^
  23. ^
  24. ^
  25. ^
  26. ^
  27. ^
  28. ^
  29. ^
  30. ^
  31. ^
  32. ^ a b c d
  33. ^
  34. ^
  35. ^
  36. ^ .
  37. ^
  38. ^ a b
  39. ^
  40. ^
  41. ^
  42. ^
  43. ^
  44. ^
  45. ^
  46. ^
  47. ^
  48. ^ a b
  49. ^
  50. ^ Deeks, S. G.; & McCune, J. M. Can HIV be cured with stem cell therapy?. Nature Biotechnology, 2010, 28(8), pp 807.
  51. ^ Alkhatib, G. The biology of CCR5 and CXCR4. Current Opinion in HIV and AIDS, 2009, 4(2), pp 96.
  52. ^ a b Hütter, G.; Nowak, D.; Mossner, M.; Ganepola, S.; Müßig, A.; Allers, K.; ... & Thiel, E. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. New England Journal of Medicine, 2009, 360(7), pp 692-698.
  53. ^ Carroll, D. Progress and prospects: zinc-finger nucleases as gene therapy agents. Gene Therapy, 2008, 15(22), pp 1463-1468.
  54. ^ Perez, E. E., Wang, J., Miller, J. C., Jouvenot, Y., Kim, K. A., Liu, O., ... & June, C. H.. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nature biotechnology, (2008) 26(7), pp 808-816.
  55. ^ Chung, J., Rossi, J. J., & Jung, U.. Current progress and challenges in HIV gene therapy. Future virology, (2011) 6(11), pp 1319-1328.
  56. ^ a b Lai, Y. CCR5-targeted hematopoietic stem cell gene approaches for HIV disease: Current progress and future prospects Current Stem Cell Research and Therapy, (2012) 7 (4), pp. 310-317.
  57. ^ a b
  58. ^ a b c d e Urnov, F. D.; Rebar, E. J.; Holmes, M. C.; Zhang, H. S.; & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 2010, 11(9), pp 636-646.
  59. ^ a b Urnov, F. D.; Miller, J. C., Lee; Y. L., Beausejour; C. M., Rock, J. M.; Augustus, S.; ... & Holmes, M. C.. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, (2005), 435(7042), pp 646-651.
  60. ^ Wayengera, M.Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy.Theor Biol Med Model, (2011), 8, pp26.
  61. ^ Wayengera, M.Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia.Theor Biol Med Model, (2011), 9, pp30.
  62. ^ Wayengera, M.Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/therapy precursors.Theor Biol Med Model, (2011), 8, pp23.
  63. ^ Wayengera M: HIV and Gene Therapy: The proposed [R-M enzymatic] model for a gene therapy against HIV. Makerere Med J. 2003, 38:28-30.
  64. ^ Wayengera M, Kajumbula H, Byarugaba W: Frequency and site mapping of HIV-1/SIVcpz, HIV-2/SIVsmm and Other SIV gene sequence cleavage by various bacteria restriction enzymes: Precursors for a novel HIV inhibitory product. Afr J Biotechnol 2007, 6(10):1225-1232.
  65. ^ Wayengera M, Kajumbula H, Byarugaba W: Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: inherent potential for biosynthetic versus live microbicides. Theor Biol Med Model. 2008, 5:18.
  66. ^ Wayengera M: Pre-Integration gene slicing (PRINT-GSX) as an alternate or complimentary gene therapy modem to RNA interference. J Appl Biol Sci. 2008, 1(2):56-63.
  67. ^ Wayengera M: Diverting primary HIV entry and replication to vaginal commensal lactobacillus expressing R-M nucleic enzymatic peptides with potent activity at cleaving proviral DNA as a novel HIV live microbicide strategy. Microbicide- New Delhi, India 2008. Abs-10.
  68. ^ Wayengera M: Preparing for a Phase 1 Preclinical trial of VRX-SMR: a Lentiviral Vector transduced with restriction enzymes cleaving HIV proviral DNA as a therapeutic vaccine: Opportunities and Challenges. Vaccine Congress -Amsterdam, Netherlands 2007, :24OR.
  69. ^ Wayengera M: xREPLAB: A recombinant lactobacillus strain producing restriction enzymes with potent activity against HIV proviral DNA as a Live Microbicide Strategy. AIDS vaccine- Washington, Seattle 2007, :P05-01.
  70. ^ Wayengera M: A Recombinant lactobacillus strain producing restriction enzymes that cleave proviral HIV DNA may offer a novel Vagino-cervical mucosal exogenous live Microbicide strategy for preventing HIV transmission among high-risk women. Afr J Biotechnol 2007, 6(15):1750-1756.
  71. ^ Wayengera M: PREX-1979: Modeling the first ever prototype of could be a 5th generation of Microbicides for preventing HIV infection among high-risk women. Afr J Biotechnol. 2007, 6(10):1221-1224.
  72. ^ Wayengera M: Why bacteria derived R-M nucleic enzymatic peptides are efficient therapeutic molecules for use in the design and development of novel HIV therapeutic molecules. Afr J Biotechnol. 2008, 7 (12): 1791–1796.
  73. ^ a b
  74. ^ a b c d e f g h i j k Pavletich, N. P.; & Pabo, C. O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science, 1991, 252(5007), pp 809-817.
  75. ^ a b c d e f g h i j k Klug, A.. The discovery of zinc fingers and their development for practical applications in gene regulation. Proceedings of the Japan Academy, Series B, (2005)81(4), pp 87-102.
  76. ^ Frankel, A. D.; Berg, J. M.; & Pabo, C. O. Metal-dependent folding of a single zinc finger from transcription factor IIIA. Proceedings of the National Academy of Sciences, 1987, 84(14), pp 4841-4845.
  77. ^ a b Lee, M. S.; Gippert, G. P.; Soman, K. V.; Case, D. A.; & Wright, P. E. Three-dimensional solution structure of a single zinc finger DNA-binding domain. Science, 1989, 245(4918), pp 635-637.
  78. ^ a b c d Klug, A.; & Schwabe, J. W. Protein motifs 5. Zinc fingers. The FASEB Journal, 1995, 9(8), pp 597-604.
  79. ^ a b c d Stone, D.; Kiem, H. P.; & Jerome, K. R. Targeted gene disruption to cure HIV. Curr Opin HIV AIDS. 2013, 8, pp 000-000.
  80. ^ a b Coakley, E., Petropoulos, C.J., Whitcomb, J.M. Assessing ch vbgemokine co-receptor usage in HIV. Curr. Opin. Infect. Dis. (2005) 18(1), pp 9-15.
  81. ^ Wilen, C.B., Wang, J., Tilton, J.C., et al. Engineering HIV-resistant humant CD4+ T cells with CXCR4-specific zinc-finger nucleases. PLoS Pathogens. (2011)
  82. ^ Didigu, C.A., Wilen, C.B., Wang, J. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood (2013)
  83. ^ a b Barton, K. M.; Burch, B. D.; Soriano-Sarabia, N.; & Margolis, D. M. Prospects for treatment of latent HIV. Clinical Pharmacology & Therapeutics. 2012.
  84. ^ a b c d e f Cathomen, T., & Joung, J. K.. Zinc-finger nucleases: the next generation emerges. Molecular Therapy, (2008) 16(7), pp 1200-1207.
  85. ^ a b Levine, B. L.; Humeau, L. M.; Boyer, J.; MacGregor, R. R.; Rebello, T.; Lu, X.; ... & June, C. H. "Gene transfer in humans using a conditionally replicating lentiviral vector". Proceedings of the National Academy of Sciences, (2006), 103(46), pp 17372-17377.
  86. ^ Varela-Rohena, A., Carpenito, C., Perez, E. E., Richardson, M., Parry, R. V., Milone, M., ... & Riley, J. L. "Genetic engineering of T cells for adoptive immunotherapy". Immunologic research, (2008) 42(1-3), pp 166-181.
  87. ^ a b c d e Rosenberg, T. "The man who had HIV and now does not". New York Magazine. Retrieved January 2013.
  88. ^


See also

Timothy Ray Brown, who underwent a bone marrow transplant in 2007 to treat leukemia, had HIV simultaneously.[87] Soon after the operation the HIV dropped to undetectable levels.[87] This is a result of the bone marrow donor being homozygous for the CCR5-Δ32 mutation.[87] This new mutation conferred a resistance to HIV in the recipient, eventually leading to an almost complete disappearance of HIV particles in his body.[87] After nearly 2 years without antiretroviral drug therapy, HIV could still not be detected in any of his tissues.[87][88] Though this method has been effective at reducing the level of infection, the risks associated with bone marrow transplants outweighs its potential value as a treatment for HIV.[52]

The Berlin Patient

The cells in which the mutations are induced ex vivo are filtered out from lymphocytes by apheresis to produce analogous lentiviral engineered CD4+ T-cells.[85] These are re-infused into the body as a single dose of 1 X 1010 gene modified analogous CD4+ T-cells.[85] A viral vector is used to deliver the ZFNs that will induce the desired mutation into the cells. Conditions that promote this process are carefully monitored ensuring the production of CCR5 strain HIV-resistant T cells.[86]

Administration of treatment

To employ the ZFNs in clinical settings the following criteria need to be met: i) High specificity of DNA-binding – Correlates with better performance and less toxicity of ZFNs. Engineered ZFNs take into account positional and context-dependent effects of zinc fingers to increase specificity.[84] ii) Enable allosteric activation of FokI once bound to DNA in order for it to produce only the required DSB.[84] iii) In order to deliver two different zinc finger nuclease subunits and donor DNA to the cell, the vectors that are used need to be improved to decrease the risk of mutagenesis.[84] These include adeno-associated virus vectors, integrase-deficient lentiviral vectors and adenovirus type 5 vectors.[84] iv) Transient expression of ZFNs would be preferred over permanent expression of these proteins in order to avoid ‘off-target’ effects.[84] v) During gene targeting, genotoxicity associated with high expression of ZFNs might lead to cell apoptosis and thus needs to be thoroughly verified in vitro and in vivo transformation assays.[84]

Several issues exist with this experimental treatment. One issue lies in ensuring that the desired repair mechanism is the one that is used to repair the DSB following gene addition.[83] Another issue with the disruption of the CCR5 gene is that CXCR4-specific or dual-tropic strains are still able to access the cell.[83] This method can prevent the progression of HIV infection.

Current ZFN treatments focus on the CCR5 gene as no known side effects result from altering CCR5.[80] There are strains of HIV that are able to use CXCR4 to enter the host cell, bypassing CCR5 altogether.[80] The same gene editing technology has been applied to CXCR4 alone and in combination with CCR5 [81][82]

Increasing the number of zinc fingers increases the specificity by increasing the number of base pairs that the ZFN can bind to.[4] However too many zinc fingers can lead to off-target binding and thus offsite cleavage.[4] This is due to an increased likelihood of zinc fingers binding to parts of the genome outside of the gene of interest.


The main DSB repair pathway in mammals (that occurs in the absence of a homologous nucleotide base sequence that can be used by a homologous recombination mechanism is through non-homologous end joining (NHEJ).[79] NHEJ, although capable of restoring a damaged gene, is error-prone.[79] DSB are, therefore, introduced into the gene until an error in its repair occurs at which point ZFNs are no longer able to bind and dimerize and the mutation is complete.[79] In order to accelerate this process, exonucleases can be introduced to digest the ends of the strands generated at DSBs.[79]

Introducing genome alterations depends upon either of the two natural repair mechanisms of a cell: non-homologous end joining (NHEJ) and homology-directed repair (HDR).[58] Repair through NHEJ comes about by the ligation of the end of the broken strands and, upon the occurrence of an error, can produce small insertions and deletions. HDR, on the other hand, makes use of a homologous DNA strand in order to repair and gene and making use of this repair mechanism and providing the desired nucleotide sequence allows for gene insertion or modification.[58]

Introducing the CCR5 mutation

The dimerization of two ZFNs is required to produce the necessary double-strand break within the CCR5 gene because the interaction between the FokI enzyme and DNA is weak.[58] This break is repaired by the natural repair mechanisms of the cell, specifically non-homologous end joining.[58]

Specificity is provided by dimerization, which decreases the probability of off-site cleavage. Each set of zinc fingers is specific to a nucleotide sequence on either side of the targeted gene 5-7 bp separation between nuclease components.[4]

It has been discovered that FokI has no intrinsic specificity in its cleavage of DNA and that the zinc finger recognition domain confers selectivity to zinc finger nucleases.[4][59]

Nuclease dimerization and cleavage

Also serine 75 on the third finger hydrogen bonds to the phosphate between base pairs 7 and 8, as the only backbone contact with the secondary strand of DNA.[74][75][78]

In addition to histidine, a conserved arginine on the second beta strand of the zinc fingers makes contact with the phosphodiester oxygen on the DNA strand.[74][75][78]

The histidine coordinated to the zinc atom, which is also the seventh residue in the alpha helix of the zinc fingers, coordinates the Zinc ion through its Nε and hydrogen bonds with phosphodiester oxygen through Nδ on the primary DNA strand.[74][75][78]

Contacts with DNA backbone

At the 6th residue, fingers 1 and 3 have arginine donating a pair of charged hydrogen bonds to Nitrogen 7 and Oxygen 6 of guanine at the 5’ end enhancing the site recognition sequence of zinc fingers.[74][75]

In residue 3 of the 2nd (i.e., middle) zinc finger, histidine49 forms a hydrogen bond with a co-planar guanine in base pair 6. The stacking of Histidine against Thymine in base pair 5 limits the conformational ability of Histidine49 leading to increased specificity for the histidine-guanine hydrogen bond.[74][75]

Each zinc finger has an arginine (arg) amino acid protruding from the alpha helix, which forms a hydrogen bond with Nitrogen 7 and Oxygen 6 of the guanine (gua) that is located at the 3’ end of the binding site.[74][75][77] The arg-gua bond is stabilized by aspartic acid from a 2nd residue, which positions the long chain of arginine through a hydrogen bond salt bridge interaction.[74][78]

The zinc atom is constrained in a tetrahedral conformation through the coordination of Cys3, Cys6, His19, and His23 and Zinc – Sulfur bond distance of 2.30 +/- 0.05 Angstroms and Zinc – Nitrogen bond distances of 2.0 +/- 0.05 Angstroms.[75][76][77]

Zinc fingers are repeated structural protein motifs with DNA recognition function that fit in the major grooves of DNA.[74] Three zinc fingers are positioned in a semi-circular or C-shaped arrangement.[75] Each zinc finger is made up of anti-parallel beta sheets and an alpha helix, held together by a zinc ion and hydrophobic residues.[74][75]

The amino terminus of the alpha helix portion of zinc fingers targets the major grooves of the DNA helix and binds near the CCR5 gene positioning FokI in a suitable location for DNA cleavage.[4][74][75]

The exact constitution of the ZFNs that are to be used to treat HIV is still unknown. The binding of ZFNs for the alteration of the Zif268 genelink, however, has been well-studied and is outlined below in order to illustrate the mechanism by which the zinc finger domain of ZFNs bind to DNA.[74][75]

The FokI catalytic domain must dimerize in order to cleave the DNA at the targeted site, and requires there to be two adjacent zinc finger nucleases (see picture), which independently bind to a specific codon at the correct orientation and spacing. As a result, the two binding events from the two zinc finger nuclease enables specific DNA targeting.[73] Specificity of genome editing is important in order for the zinc finger nuclease to be a successful application. The consequence of off-targeting cleavage can lead to a decrease in efficiency of the on-target modification in addition to other unwanted changes.[73]

Zinc finger binding

A recent and unique application of ZFN-technology to treat HIV has emerged whose focus is to target not the host genome, but rather proviral HIV DNA, for mutagenesis.[60] The authors of this work have drawn their inspiration from the innate defense mechanism against bacteria-infecting-viruses called bacteriophages, present amongst those bacteria endowed with restriction modification (R-M) systems. These bacteria secrete a restriction enzyme (REase) that recognizes and repetitively cleaves around palindromic sequences within the xenogenic DNAs of the bacteriophages or simply phages, until the same is disabled. Further support for this approach resides in the fact that, the human genome comprises in large part remnants of retroviral genomes that have been inactivated by several mechanisms, some of whose action resembles that of ZFN. It should not be surprising, therefore, that the initial work leading to the application of ZFN technology in this manner revolved around and involved the isolation and testing of HIV/SIV targeting bacteria-derived REases, whose non-specificity (due to their short recognition sequences) unfortunately, rendered them toxic to the host genome. The latter-potential host-genome toxicity posed by the raw bacteria-derived REases limited their application to ex-vivo modalities for HIV prevention, namely synthetic or live microbicides. Subsequently, however, the unique specificity offered by ZFNs was quickly recognized and harnessed, paving way for a novel strategy for attacking HIV in-vivo (through target mutagenesis of proviral HIV DNA) that is similar to the manner by which bacteria equipped with R-M systems do, to disable the foreign DNAs of in-coming phage-genomes. Because latent proviral HIV DNA resident in resting memory CD4 cells forms the major barrier to the eradication of HIV by highly active antiviral therapy (HAART), it is speculated that this approach may offer a 'functional cure" for HIV. Both ex-vivo (manipulation of stem or autologous T cell precursors) and in-vivo delivery platforms are being explored. It is also hoped that, when applied to non-HIV infected persons, this strategy could offer a genomic vaccine against HIV and other viruses. Similar work is ongoing for high-risk HPVs (with the intent of reversing cervical neoplasia) [61] as well as with HSV-2 (with the goal of achieving a complete cure for genital herpes) [62][63][64][65][66][67][68][69][70][71][72]

Proviral HIV DNA

The zinc finger nucleases that have been synthesized for this treatment are manufactured by combining FokI Type II restriction endonucleases with engineered zinc fingers.[4][59] The number of zinc fingers attached to the endonuclease controls the specificity of the ZFN since they are engineered to preferentially bind to specific base sequences in DNA. Each ZFN is made up of multiple zinc fingers and one nuclease enzyme.[4]

It has also been observed that 20% of the Caucasian population possess a mutation, called CCR5-Δ32 (frequency of 0.0808 for homozygous allele), that prevents the CCR5 chemokine receptor protein, which is the main means of viral access into the cell, from being expressed on the surface of their CD4+ T-cells.[51][52][53][54][55] Individuals who are homozygous for this mutation are immune to HIV strains that utilize the CCR5 receptor in order to gain access to the cell while those who are heterozygous for this mutation have been found to have reduced plasma viral load in addition to a delayed progression to AIDS.[56][57] By combining these facts, researchers have proposed a novel method of treatment for HIV. This method attempts to treat the infection by disrupting the CCR5 gene, such as introducing the CCR5-Δ32 mutation using a recombinant adenoviral vector or forcing DNA repair by nonhomologous end joining, which is prone to error and results in a non-functional gene. As a consequence, resulting in the expression of nonfunctional CCR5 co-receptors on CD4+ T cells, providing immunity against infection.[4][56][57][58]

Host cellular co-receptors for HIV

Since antiretroviral therapy requires a lifelong treatment regimen, research to find more permanent cures for HIV infection is currently underway.[50] It is possible to synthesize zinc finger nucleotides with zinc finger components that selectively (almost selectively) bind to specific portions of DNA. Conceptually, targeting and editing could focus on host cellular co-receptors for HIV or on proviral HIV DNA.

Zinc finger nuclease treatment of HIV

Zinc-finger nickases (ZFNickases) are created by inactivating the catalytic activity of one ZFN monomer in the ZFN dimer required for double-strand cleavage.[48] ZFNickases demonstrate strand-specific nicking activity in vitro and thus provide for highly specific single-strand breaks in DNA.[48] These SSBs undergo the same cellular mechanisms for DNA that ZFNs exploit, but they show a significantly reduced frequency of mutagenic NHEJ repairs at their target nicking site. This reduction provides a bias for HR-mediated gene modifications. ZFNickases can induce targeted HR in cultured human and livestock cells, although at lower levels than corresponding ZFNs from which they were derived because nicks can be repaired without genetic alteration.[38][49] A major limitation of ZFN-mediated gene modifications is the competition between NHEJ and HR repair pathways. Regardless of the presence of a DNA donor construct, both repair mechanisms can be activated following DSBs induced by ZFNs. Thus, ZFNickases is the first plausible attempt at engineering a method to favor the HR method of DNA repair as opposed to the error-prone NHEJ repair. By reducing NHEJ repairs, ZFNickases can thereby reduce the spectrum of unwanted off-target alterations. The ease by which ZFNickases can be derive from ZFNs provides a great platform for further studies regarding the optimization of ZFNickases and possibly increasing their levels of targeted HR while still maintain their reduced NHEJ frequency.

Zinc-finger nickases


This article was sourced from Creative Commons Attribution-ShareAlike License; additional terms may apply. World Heritage Encyclopedia content is assembled from numerous content providers, Open Access Publishing, and in compliance with The Fair Access to Science and Technology Research Act (FASTR), Wikimedia Foundation, Inc., Public Library of Science, The Encyclopedia of Life, Open Book Publishers (OBP), PubMed, U.S. National Library of Medicine, National Center for Biotechnology Information, U.S. National Library of Medicine, National Institutes of Health (NIH), U.S. Department of Health & Human Services, and, which sources content from all federal, state, local, tribal, and territorial government publication portals (.gov, .mil, .edu). Funding for and content contributors is made possible from the U.S. Congress, E-Government Act of 2002.
Crowd sourced content that is contributed to World Heritage Encyclopedia is peer reviewed and edited by our editorial staff to ensure quality scholarly research articles.
By using this site, you agree to the Terms of Use and Privacy Policy. World Heritage Encyclopedia™ is a registered trademark of the World Public Library Association, a non-profit organization.

Copyright © World Library Foundation. All rights reserved. eBooks from Project Gutenberg are sponsored by the World Library Foundation,
a 501c(4) Member's Support Non-Profit Organization, and is NOT affiliated with any governmental agency or department.