Zinc finger nuclease--a new knock-out technique

Why zinc finger nuclease in zebrafish?

• 1. There is no effectvie reverse genetic tool for targeted knockouts in zebrafish.

• 2. RNAi and morpholino come with substantial technical limitation.

What is a Zinc Finger Nuclease (ZFN)?

Stephen C. Ekker, 2008

How ZFN works?

http://labs.umassmed.edu/WolfeLab/danio_rerio.html

Oligomerized Pool ENgineering

Zinc Finger Engineering

combinatorial-based selection method--

How can we use ZFN?

Advantage of Zinc Finger Nuclease

• 1. Highly specific

• 2. Available in many organism: zebrafish, human, rat...

• 3. High efficiency (compared to homologous recombination)

Summary

• 1. Zinc finger nuclease consists of Zinc finger targeting specific DNA sequences and FokI for cleavage of DNA.

• 2. Zinc finger nuclease is powerful Knock-out technique:

-specific target to the gene of interest

-high efficiency

Thank You!

Why are these sometimes referred to as ZFN pairs?A pair of ZFNs is required to cleave double-stranded DNA. This is a requirement of the FokI nuclease. FokI must dimerize to achieve a double strand break in the DNA. You will be provided with a pair of ZFNs for your target site that has been validated to cut at the endogenous chromosomal locus in a proxy cell line.

What specificity should be expected from a ZFN?Due to the dimerization requirement of the FokI endonuclease, a pair of ZFNs is required to cause a double strand break. This strategy requires two different ZFNs to bind at the target site. Each ZFN recognizes a different 12-18 base pair target sequence, and these target sequences must be separated by 4-7 base pairs to allow formation of the catalytically active FokI dimer. These positional constraints drive a very high degree of specifi city.

How is mutation induced? -non-homologous end joining

double strand breakage

non-homologous end joining template

mutation induced

FokIFokI

FokI

What are the advantages to delivering ZFNs to cells in mRNA form?There are many advantages to using a ZFN mRNA transcript. We have found the following benefits in a number of tested cell types:

1. Eliminates the risk of random genome integration of the expression plasmid DNA. 2. Lower cytotoxicity (RNA vs. DNA). 3. Higher efficiency in most tested cases. 4. Expanded range of cell types that zinc finger nucleases can be applied to because some cell types do not tolerate input DNA. 5. Eliminates the need to use different promoters for ZFN expression in certain cell types. RNA is universal to all cell types. 6. Eliminates the necessity of nuclear delivery, allowing a larger range of transfection reagents to be usable in delivery. Expression vectors have to enter the nucleus to be transcribed, while mRNA gets translated in the cytoplasm. 7. Lower off-target events by exposing cells to ZFNs for a shorter time (mRNA has a shorter half life than DNA).

What is zinc finger protein?

PD Doland, 2006

Zinc Finger Protein-the most abundant DNA binding motif

Tupler, 2001

Early approaches of genome editing

• 1. genome-wide, non-targeted approaches -radiation -chemical induced mutagenesis

• 2. conventional gene targeting -homologous recombination -low efficiency (sophisticated strategy)