zinc finger nuclease technology

7/28/2019 ZINC FINGER NUCLEASE TECHNOLOGY

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PRIYESH V WAGHMARE


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NEED? Microinjection and homologous recombination in

embryonic stem (ES) cells, are robust but overallinefficient.

Only a few percent of the injected eggs giving rise totransgenic animals.

sperm-mediated gene transfer remain poorly used

alternative strategies to the classical transgenicmethods.


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What Is ZFN Technology?

Engineered DNA-binding proteins.

Highly targeted double-strand break (DSB) withinthe genome.

Manipulation of the genome. Unprecedented ease and precision.

Double-strand breaks in DNA at user-specifiedlocations.

Double-strand breaks are important for site-specific mutagenesis.

Stimulate the cell's natural DNA-repair processes.


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What are Zinc Finger Nucleases?

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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 aZinc Finger Protein, each with specificity of 24 bp.

Highly-specific pair of 'genomic scissors' are created.


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MECHANISM


ZFN-Mediated Targeted Genome Editing


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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 theuser-defined locus.

Living cells have evolved several methods to repairdouble-strand breaks.

Endogenous processes can be harnessed to create geneknockouts or knock-ins.


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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


9/19Provost et al.


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Recent Developments in Animals

Highly effective not only in cell lines, but also inembryos for the creation of animal models.

Proven to work in a wide variety of organismsincluding 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 oforganisms.


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Benefits: Unlimited Species Possibilities

Animals with ES cell method limitations can now be targeted

Rapid Animal EngineeringFastest method for creation of knockout rodents (2-3 months) and

other higher eukaryotes

Robust Mutation RateAchieve up to a 10-15% mutation rate in founder animals

Heritable TransmissionFaithful germline transmission of targeted mutations

Universal ToolMove quickly from cell line proof-of-concept studies into animals


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ZFN Knockout Animal Creation via Microinjection



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Using ZFNs to Create Modified Cell Lines



14/19Provost et al.


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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 ofproteins or antibodies


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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 ZFNactivity in many other cell types.

CompoZr ZFNs

CompoZr Targeted Integration Kit - AAVS1

- Rapid Gene Insertion into the Human Cell Lineof Your Choice


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CONCLUSION

Highly attractive alternative to ES cell manipulationand nuclear transfer technology.

Development of large animal models for humandiseases 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.


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REFERENCES Zinc finger nuclease technology heralds a new era in

mammalian transgenesis.

BY- Fabienne Le Provost1, Simon Lillico2, BrunoPasset, Rachel Young, Bruce Whitelaw and Jean-Luc

Vilotte.

http://www.sigmaaldrich.com/life-science/zinc-

finger-nuclease-technology
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zinc finger nuclease technology

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