Zinc finger nuclease in plants

Advanced Plant Molecular BiologyHuong T. Tran

2015.05.14

PLANT MOLECULAR GENETICS LAB

Contents

1. Introduction - Zinc finger nuclease2. Structure of zinc finger nuclease3. Zinc finger fuction in plants

4. Summary

INTRODUCTION

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- ZFPs is a synthetic restriction enzymes that can be specifically designed to cleave virtually any long stretch of double-stranded DNA sequence.

What Is Zinc Finger Nuclease ?

http://www.sigmaaldrich.com

- Delivered to the cell as DNA and RNA- ZFN protein 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 knock-outs or knock-ins

StructureEach ZFN consits two functional domains:

1. DNA binding domain: comprised of a chain of two-finger modules

2. DNA cleaving domain: comprised of the nuclease domain of FokI restriction enzyme

- Recognizing a unique hexamer ( 6bp) sequence of DNA

- Two- finger modules are stitched together to form a Zinc Finger Protein, each with specificity of >

24bp

- Highly- specific of “genomic scissors” are created.

http://www.nature.com/nrg/posters/zinc-fingers

Function of Zinc finger - ZFNs can be designed to bind and cleave virtually any stretch of DNA sequence, thereby

allowing for the creation of DNA double-strand breaks (DSBs) at specified loci.- ZFNs have been used to enhance DNA integration via homologous recombination (HR) and

as an efficient tool for the induction of site-specific mutagenesis in plant species.- Expression of genes encoding ZFNs and concomitant cleavage at endogenous genomic loci

has been demonstrated in a broad spectrum of organisms, including human, mammalian and plants.

Zinc Finger Nuclease in Plants

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Examples of ZFN-mediated genome editting in plants

Plant Biotechnology Journal, 2012,10, 373–389

Repairing double-strand break ( DSBs)2 pathways :

- homology-directed repair (HDR, HR): need homologous sequences as templates for synthesis.

- nonhomologous end joining (NHEJ) of broken ends: without homologous sequences, directly ligated.

Yiping Qi et al. Genome Res. 2013;23:547-554

A pathway choice model for repair of a DNA DSB in Arabidopsis.

MH: microhomology

Targeted genome modification via double-strand break (DSB) repair.

In Vitro Cell.Dev.Biol.—Plant (2015) 51:1–8

Repairing double-strand break ( DSBs)

With Donor DNAWithout Donor DNA

Strategies for novel restriction enzymes (NRE)-mediated genome editing in plants.

ZFN-Mediated Targeted Mutagenesis

Plant Biotechnology Journal, 2012,10, 373–389

Sequence modification of preintegrated reporter constructs

- ZFN can be used for make targeted mutagenesis of preintegrated sequences in plant

- ZFN cleavage site within a preintegrated reporter gene has been used to study targeted mutagenesis

- Preintegrating a reporter constructs containing a nonfunctional herbicide resistance gene flanked by ZFN

binding site result in a locus capable of being targeted

Plant Biotechnology Journal, 7, 2009, 821–835

ZFN in make sequence modification of preintegrated reporter constructs.

Sequence modification of endogenous genes.

- Expression of genes encoding ZFNs can generate heritable mutations at targeted endogenous loci- ZFNs can efficiently cleave and stimulate mutations at an endogenous target gene in Arabidopsis- ZFNs can form the basis of a highly efficient method for site-directed mutagenesis of higher plant genes

Consensus ZFN target sites in the Arabidopsis ABI4 gene.

Keishi Osakabe et al. PNAS 2010;107:12034-12039

ZFN target sites in the ADH1 and TT4 coding sequences

Zhang et al, PNAS – 6th 29, 2010, 107

Gene deletion

- ZFN-mediated cleavage can result in larger DNA sequence deletions- The advantage of ZFNs for targeted genome modification is that, they can be designed to cleave any DNA

sequence thereby expanding the range of sequences that can be deleted..- >>> ZFN is a potentially powerful means of creating targeted genome sequence deletions

In Vitro Cell.Dev.Biol.—Plant (2015) 51:1–8

Gene editing using HDR

- HDR: homologous directly repair, need a donor template DNA for double strand break repair- Donor template DNA: contains 750- 1000-kb stretches of sequence homologous to that flanking the genomic

cleavage site and the desired sequence modification. - the frequency of HDR in somatic plant cells appears to be extremely low, so the identification and isolation of

such modifications can be a challenge- One of the future challenges for gene editing via HDR will involve the targeted modification of endogenous

genes without selectable phenotypes.

Plant Biotechnol J. 2013 May;11(4):510-5

Overview of ZFN and HDR1 example for HDR in Arabidopsis

Targeted Transgene Integration

- Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research.

- designed ZFNs can drive site-directed DNA integration into transgenic and native gene loci

- Using reporter gene: GUS , GFP reporters - i.e. : Preintegrated loci have also been

successfully targeted using NHEJ. In this system, identical ZFN cleavage sites were present in the genome and on the donor template DNA.

Targeted integration into preintegrated loci.

Plant Biotechnology Journal, 10, 373–389

A set of in-planta ZFN validation assays and their vector systems.

Targeted integration into endogenous loci

- ZFNs designed to cleave endogenous genomic sequences have been used to facilitate site-specific transgene integration into native genes. - i.e.: genes encoding ZFNs designed to cleave in exon 2 of the maize IPK1 gene were co-delivered with donor template DNA containing a promoterless PAT gene with a 2A ‘stutter’ sequence flanked by 815 bp of sequence homologous to IPK1. Targeted cleavage at the IPK1 locus and precise ‘trapping’ of the IPK1 promoter resulted in site-specific integration and herbicide resistance

VK Shukla et al. Nature 000, 1-5 (2009)

Targeting in DNA Repair Mutants

- There are 2 pathways to DNA repair: HDR vs NHEJ. - Genes encoding ZFNs have been delivered into various genetic backgrounds in which specific genes involved

in DNA repair have been mutated. - Manipulating DNA repair pathways provides a level of control over the types of sequence modifications

resulting from ZFN-mediated cleavage and a potential path to increased mutagenesis and gene targeting

frequencies.

Genome Res, 2013 23:547–554

Summary- The use of ZFNs to make targeted DSBs has made a major impact on two broad areas of plant biology. 1st : functional genomics > more effective hypothesis-driven gene discovery efforts for novel trait development. 2nd : in application major: applied crop improvement and commercial product development.

A pair of ZFNs designed to target a genomic sequence will make a break in both DNA strands specifically at that site. DNA DSBs are potentially lethal and therefore cells have several pathways for repairing breaks and restore intact DNA. There are two major DSB repair pathways: copying of information from the corresponding chromosome by HR, or simply joining the broken ends in a process called NHEJ, which often results in new mutations at the target sequence. These can include small or large deletions and insertions, or base substitutions

Arabidopsis thalianaThis small flowering plant is a key model species owing to its comparatively simple genome and rapid development. Several groups have achieved targeted disruptions of endogenous genes at frequencies of a few per cent

AgricultureZFNs have been used for gene disruption and gene replacement in cultures of maize and tobacco. Both species can be regenerated from such cultures, which facilitates delivery of ZFNs and characterization of sequence changes.The limitation in applying this approach to other species will be methods for delivery. Plants with directed changes in endogenous genes might be more publicly acceptable than the current genetically modified crops

Gene targeting Applications

http://www.nature.com/nrg/posters/zinc-fingers

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References

1. Joseph F. Petolino, 2015, Genome editing in plants via designed zinc finger nucleases, In Vitro

Cell.Dev.Biol.—Plant (2015) 51:1–8.

2. de Pater S, Pinas JE, Hooykaas PJ, van der Zaal BJ2013. ZFN-mediated gene targeting of the Arabidopsis

protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation, Plant

Biotechnol J. 2013 May;11(4):510-5

3. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I,Beausejour CM, Waite AJ, Wang NS, Kim

KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture

for highly specific genome editing. Nat Biotechnol 25:778–785

4. Osakabe K, Osakabe Y, Toki S (2010) Site-directed mutagenesis in Arabidopsis using custom-designed zinc

finger nucleases. Proc Natl Acad Sci U S A 107:12034–12039.

5. Qi Y, Zhang Y, Zhang F, Baller JA, Cleland SC, Ryu Y, Starker CG, Voytas DF (2013) Increasing frequencies of

site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways.

Genome Res 23:547–554.

6. Weinthal DM, Taylor RA, Tzfira T (2013) Nonhomologous end joiningmediated gene replacement in plant

cells. Plant Physiol 162:390–400

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