ZINC FINGER NUCLEASE TECHNOLOGY

1. PRIYESH V WAGHMARE

2. NEED?
 Microinjection and homologous recombination in
embryonic stem (ES) cells, are robust but overall
inefficient.
 Only a few percent of the injected eggs giving rise to
transgenic animals.
 sperm-mediated gene transfer remain poorly used
alternative strategies to the classical transgenic
methods.

3. What Is ZFN Technology?
 Engineered DNA-binding proteins.
 Highly targeted double-strand break (DSB) within
the genome.
 Manipulation of the genome.
 Unprecedented ease and precision.
 Double-strand breaks in DNA at user-specified
locations.
 Double-strand breaks are important for site-
specific mutagenesis.
 Stimulate the cell's natural DNA-repair processes.

4. What are Zinc Finger Nucleases?
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5.  Each Zinc Finger Nuclease (ZFN) consists of two
functional domains:
a] A DNA-binding domain comprised of a chain of two-
finger modules.
b] A DNA-cleaving domain comprised of the nuclease
domain of Fok I.
 Recognizing a unique hexamer (6 bp) sequence of
DNA.
 Two-finger modules are stitched together to form a
Zinc Finger Protein, each with specificity of ≥ 24 bp.
 Highly-specific pair of 'genomic scissors' are created.

6. MECHANISM
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ZFN-Mediated Targeted Genome Editing

7.  Designed to target any gene in any genome.
 Delivered to the cell as DNA or RNA.
 ZFN proteins are expressed.
 Translocate to the nucleus.
 Bind their target sites with high specificity.
 FokI nuclease forms its catalytically active dimer.
 Creates a single, specific double-strand break at the
user-defined locus.
 Living cells have evolved several methods to repair
double-strand breaks.
 Endogenous processes can be harnessed to create gene
knockouts or knock-ins.

8. REPAIR:
 Non-homologous end joining (NHEJ).
 Homologous recombination (HR).
 NHEJ is an imperfect repair system
-Insertions or deletions of base pairs.
-Creation of a frameshift.
-Exon skipping.
-Disrupt gene translation.
-Knockout gene function.
 Donor plasmid.
-Donor for homology directed repair.
-Designed to include transgenes for targeted integration

9. Provost et al.

10. Recent Developments in Animals
 Highly effective not only in cell lines, but also in
embryos for the creation of animal models.
 Proven to work in a wide variety of organisms
including rats, mice, rabbits, zebrafish, Drosophila
and C. elegans.
 Does not require the use of embryonic stem (ES) cells.
 Injected directly into early stage embryos.
 Targeted gene disruption in a wider spectrum of
organisms.

11. Benefits:
 Unlimited Species Possibilities—
Animals with ES cell method limitations can now be targeted
 Rapid Animal Engineering—
Fastest method for creation of knockout rodents (2-3 months) and
other higher eukaryotes
 Robust Mutation Rate—
Achieve up to a 10-15% mutation rate in founder animals
 Heritable Transmission—
Faithful germline transmission of targeted mutations
 Universal Tool—
Move quickly from cell line proof-of-concept studies into animals

12. ZFN Knockout Animal Creation via Microinjection
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13. Using ZFNs to Create Modified Cell Lines
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14. Provost et al.

15. Target Applications
 Functional Genomics/Target Validation
 Creation of gene knockouts in multiple cell lines
 Complete knockout of genes not amenable to RNAi
 Cell-based screening
 Creation of knock-in cell lines with promoters, fusion
tags or reporters integrated into endogenous genes
 Cell Line Optimization
 Creation of cell lines that produce higher yields of
proteins or antibodies

16. Commercialization
 Sigma® has a standard offering of ZFNs for
Human, Mouse, and Rat.
 ZFNs have recently been shown to produce site-
specific gene knockouts in Zebrafish (Doyon et
al. Nature Biotechnology May 25, 2008).
 The ZFNs for this particular application were tested in
a yeast proxy system that accurately reflects ZFN
activity in many other cell types.
 CompoZr™ ZFNs
 CompoZr® Targeted Integration Kit - AAVS1
- Rapid Gene Insertion into the Human Cell Line
of Your Choice

17. CONCLUSION
 Highly attractive alternative to ES cell manipulation
and nuclear transfer technology.
 Development of large animal models for human
diseases and xeno-transplantations.
 Agricultural breeding.
 Medical research.
 Wide range of new applications in modifying the
genome of species with which it has, until
recently, remained very difficult to work.

18. REFERENCES
 Zinc finger nuclease technology heralds a new era in
mammalian transgenesis.
BY- Fabienne Le Provost1, Simon Lillico2, Bruno
Passet, Rachel Young, Bruce Whitelaw and Jean-Luc
Vilotte.
 http://www.sigmaaldrich.com/life-science/zinc-
finger-nuclease-technology