Department of Agril-biotechnology,Orissa University of Agriculture & Technology,BBSR.

Why Synthetic/ Artificial Chromosome (AC)?

Methods to develop AC

Case study

Amendments in AC

Applications

Summary Points

Future Direction

Conclusion

OUTLINES OF THE SEMINAROUTLINES OF THE SEMINAR

Need.....???

Traditional genetic engineering

1) The transformation process often results in the integration of foreign genes into an endogenous gene and disrupts its function

2) Transgenes can be influenced by upstream or downstream regulatory elements around them, that is, they are subject to position effects under these circumstances

3) Only a single gene or a few genes can be expressed

4) Most importantly, Gene stacking or pyramiding

( Halpin, 2005)

First Generation Genetic Engineering

• Gene stacking- difficult• Transgene position effects• Insertion-site complexity

Second Generation Genetic Engineering

Synthetic Chromosome

Delivery of large DNA sequences

Complete metabolic pathways

Genetic changes 100-kb to megabases (Mb)

Goyal et al., 2009

Need.....???

Artificial Chromosome:It refers to any nonintegrating vector that is transmissible and has the

ability to harbor large amounts of DNA

In bacteria – circular

In yeast, animals & plants- linear

First constructed in yeast and E. coli systems (Clarke & Carbon, 1980) Requirements for chromosomal maintenance and stability - Centromeres - Telomeres - Origins of replication

(Murray & Szostak, 1983)

Introduction

Requirement for Synthetic Plant Chromosome

Basic requirements for Synthetic Plant Chromosome

1.Centromere

2. Telomere

3. Sufficient Chromatin

4. Selectable Marker transgene

Gaeta et al., Annu. Rev. Plant Biol., 2012

Centromere Components In Plants

Centromere – Requisite component of AC

Contains tendem repeated sequences of various sizes, ~1 to 3 Mb (Copenhaver, 2003)

In grass family, there is small (156 bp) repeat sequences (Jin et al., 2004)

Study of mis-division of centromeres: The core functional B centromere is in the range of 300 kb or larger in maize (Jin et al., 2005) Retrotransposons (Neumann et al., 2009)

In maize, the tendem array size of centromere repeat C (CentC)- 700 kb to 2.7Mb but the entire length is not necessarily associated with CENH3 (Wolfgruber et al., 2009)

Epigenetic Aspects Of Centromere Specification

For construction of AC, Yeast as a model- old concept

Centromere specification in yeast is unusual among eukaryotes Centromeres in higher eukaryotes have diffuse organization (Henicoff et al., 2001)

Kinetochores form over apparently unique DNA, which is referred to as a neocentromere

Repeats are not a necessary determinant of the sites of kinetochore formation (Malik &Henikoff, 2009)

Mechanism by which centromeres are established, maintained, and function remain a mystery (Dalal, 2009)

Telomere Components In Plants

Specialized structure which cap the ends of eukaryotic chromosome

Consist of highly conserved long array of short tandemly repeated sequences e.g. TTTAGGG in A. thaliana TTAGGG in Homo sapiens TTAGG in insects

Average length: 3-40 kb on an average (Burr et al., 1992)

Functions: 1. Maintaining the structural integrity

2. Ensure complete replication of extreme ends of chromosome

Surovtseva et al., Mol. Cell, 2009

1. Yeast Artificial Chromosome (YAC)

YAC is plasmid into which yeast genes have been inserted

Developed by Murray & Szostak in 1983

Capacity- 3000 kb

YACs- Largest capacity vectors available

Advantage: Can be used to express eukaryotic proteins that require post translational modification

Disadvantages: 1. Less stable than BACs 2. Chimerism

Artificial Chromosome As Vectors

Murray and Szostak, Nature, 1983

2. Bacterial Artificial Chromosome (BAC)

Construction is based on F-plasmid

Recombinants can be identified by Lac selection

Capacity- 350 kb

Uses: 1. Extensively used to sequence the genome of organisms e.g.- HGP 2. Being utilized to a greater extent in modelling genetic diseases e.g.- Alzheimer's disease, Cancer

3. Infectious viral clones e.g.- herpesviruses, poxviruses

CONTD…CONTD…

Shizuya et al., Proc. Natl. Acad. Sci., 1992

3. Human Artificial Chromosome (HAC)

HAC is synthetically produced vector DNA, possessing the characteristics of human chromosome

Developed by Harrington et al., in 1997

Size- 1/10th to 1/5th of human chromosome- 6 to 10 Mb

Mitotically stable for up to six months

Advantage: 1. Can carry human genes that are too long

2. Can be used for gene therapy

3. Useful in expression studies as gene transfer vectors

CONTD…CONTD…

Harrington et al., Nat. Genet. 1997

Bottom-up Method

Building chromosomes by de novo assembling their component parts

This approach has been used for assembly of HAC (Harrington et al., 1997)

Upon delivery, DNA is subjected to rearrangements resulting in AC larger than the original construct (Lim & Farr, 2004)

Not yet used in plant due to high complexity & limited understanding about plant centromere organization (Houben & Schubert, 2007)

Top-down Method

Based on chromosome fragmentation or truncation

Achieved by- 1. Irradiation

2. Telomere mediated truncation

Irradiation:

Example- An unstable maize minichromosome comprising part of the short arm of chromosome 10 has been recovered as a result of pollen irradiation (Brock and Pryor, 1996)Disadvantage: Unstable

Telomere-mediated Truncation

Aim- To whittle away the chromosome arms using transformation of telomere repeats

It bypasses the complications of the epigenetic aspects of centromere specification

It works robustly in plants & can be used to produce engineered minichromosomes with endogenous centromeres

Construct- Genes of interest, Site-specific recombination cassettes, Telomere repeats (Yu et al., 2006)

B Chromosome Based Minichromosomes

We can produce A & B chromosome based minichromosomes by this method but B chromosome based minichromosome is interesting-

Reasons:- 1 Naturally occurring supernumerary chromosome

2. Basically inert, small size – no phenotype (Jones et al., 2003) 3. No developmental & transmission problem

4. Easy detection of B chromosome derivatives (Kato et al., 2005) 5. No report of recombination with A chromosome set

Minimal detrimental effect on host genome

Gaeta et al., Annu. Rev. Plant Biol., 2012

J. A. Birchler et al.,2010

J. A. Birchler et al.,2010

J. A. Birchler et al.,2010

J. A. Birchler et al.,2010

RESULTS...

Fig. 1. Chromosomal truncation constructs pWY76 and pWY86 and the control construct pWY96. Tvsp- terminator from soybean vegetative storage protein gene; Bar- bialophos resistance gene as a selection marker gene; P35S- 35S promoter from cauliflower mosaic virus; Tnos- Nos terminator from Agrobacterium; Tmas- Mas terminator from Agrobacterium; Pnos- Nos promoter from Agrobacterium; Pmas1- Mas promoter from Agrobacterium; lox and FRT, site-specific recombination sites; HPT- hygromycin B resistance gene; GFP- green fluorescent protein gene; DsRed- red fluorescent protein gene; FLP, recombinase gene; Telomeres, telomere units of pAtT4 isolated from Arabidopsis . Arrows designate the direction of transcription.

Development of Constructs

Construct Transgenic Event

Truncation locations

Pollen Abortion

pWY86 (A) B77 3 S truncation +

pWY76 (B) T87 1 S truncation +

pWY86 (C) B37 4 L truncation +

pWY86 (D) B44 4 L truncation +

Recovered transgenic lines- 126Transgenic events- 220 (T93, B83 & 44)

Transgenic Events

Pollen abortion: + = 50% pollen abortion

CONTD…

Fig. 2. Cytological detection of chromosomal truncations. Metaphase chromosomes were hybridized with pWY96 probe (red) and mixtures of repetitive sequence probes, CentC (green), knob (green), subtelomere 4–12-1 clone (green), and Cent4 (white). Arrows denote the transgene truncation sites (white arrows) and the corresponding sites on the homologues (gray arrows). (A) pWY86 ransgenic event B77 with a chromosome 3 short-arm truncation. (Inset) The chromosome 3 pair with (Left) and without (Right) the transgene (red). (B) pWY76 transgenic event T87 with a chromosome 1 short-arm terminal-knob truncation. (Inset) Chromosome 1 homologues with (Upper) and without (Lower) the transgene (red). (C and D) pWY86 transgenic events B37 and B44 with truncations of chromosome 4 long-arm terminal subtelomeric 4–12-1 sequence (green). (Insets) Chromosome 4 homologues with (Upper) and without (Lower) the transgene (red). Chromosome 4 can be identified by the Cent4 hybridization (white) at their centromeres. (Scale bar, 10 m.)

pWY86 B77 3S T pWY76 T87 1S T

pWY86 B37 4L T pWY86 B44 4L T

CONTD…

Fig. 4. Telomere sequences detected in internal chromosome locations.Metaphase chromosomes of a pWY86 transgenic line were probed with a pWY96 probe (red) and a telomere probe (green). Chromosomes were stained with DAPI. Arrowheads indicate the internal telomere (green) and transgene (red) signals. Arrowheads in the enlarged images from the top to the bottom panels denote merged transgene (red) and telomere (green) signals, and telomere only (green) and transgene only (red). (Scale bar, 10 m.)

AMENDING ENGINEERED MINICHROMOSOMES IN PLANTS

In planta Amendments

Zinc Finger NucleasesZinc Finger Nucleases

Durai et al., Nucleic Acids Res., 2005

Site-specific Recombination Systems

Cre/loxP system

Bacteriophage P1

Cre (Cyclization recombination) recombinase

loxP (locus of X-over of P1), 34-bp

Two directly repeated loxP sites - deletion

Two inverted loxP sites – inversion

Gilbertson, Trends Biotechnol., 2003

Fig-loxP Site

Inversion Deletion

CONTD…

Zinc Finger Nucleases (ZFNs)

ZFNs are artificial RE generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain from FokI

Townsend et al., Nature, 2009

It can be engineered to target desired DNA sequences

Non-specific cleavage domain: from the type IIs RE FokI is used

ZF DNA-binding domain: recognize between 9 and 18 bp

Uses: 1.Disabling an allele

2.Deletion of intervening sequences

(a) A diagrammatic engineered minichromosome generated by telomere-mediated chromosome truncation. The minichromosome contains a terminal transgene locus (red ), which contains a promoter (Pro) driving expression of a selection gene (S gene). The transgene cassette also contains a pair of directly oriented loxP sites flanking the selection gene, as well as 3’ telomere repeat sequences (Telo). Crossing a line containing this minichromosome to one that expresses Cre recombinase will lead to excision of the selection gene, and will leave a single wild-type loxP site. In a subsequent transformation, a circular construct (donor molecule 1) containing a loxP site, an attP site (PP), a promoterless S gene, a novel gene of interest (GOI-1), and two specific sites recognized by a zinc finger nuclease (ZF1) can be introduced into the minichromosome-containing cell along with a plasmid expressing Cre recombinase.

Mechanisms To Amend Minichromosome Platforms

CONTD…

(b) The first novel gene has been added to the minichromosome transgene locus as a result of the previous Cre recombination event. A specific zinc finger (targeting the ZF1 sites) could be used to delete the intervening sequences. In a second round of gene stacking, a second donor plasmid (donor molecule 2) could be introduced into cells containing the minichromosome along with an integrase enzyme that specifically leads to recombination between attP and attB sites. In this round of integration, the donor molecule contains a promoterless selection gene, a second gene of interest (GOI-2), novel attachment sites attB and attb (BB and bb, respectively), and a zinc finger nuclease recognition sequence.

Gaeta et al., Annu. Rev. Plant Biol., 2012

(c) The result of the previous integration event involves introduction of the second gene of interest. The result of recombination between attachment sites BB and PP results in the site PB, which is not acted upon by the integrase.Similarly to panel b, a zinc finger nuclease could again be used to delete extraneous sequences. A third donor cassette could be used and recombination could be targeted to the bb site (not shown). In each recombination event involving the integrase, the target sites are destroyed, and thus attB and attP could be alternately used for sequential addition to the minichromosome.

CONTD…

At any point, we can delete all sequences between the two loxP sites that flank the entire cassette by crossing this line with another line that expresses Cre recombinase

The artificial chromosome construction system can be used as powerful mutagen

(Brown et al., 2000)

It can also be used for gene stacking in plants, which is currently considered as challenging for plant biotechnology

(Halpin., 2005)

Functional genomic studies could use minichromosomes as a platform for adding specific genes or doses of chromosomal segments

Can be used in sequencing projects

POTENTIAL APPLICATIONS

AC platforms can provide solution to the stable expression & maintenance of multiple transgenes in one genome

It represents a potential powerful research tool for understanding chromosome structure & function

They can be useful for mass production of foreign proteins, pharmaceuticals, or useful metabolites in plants (Daniell et al., 2009)

The inheritance of multiple foreign genes as a unit

CONTD…

Next generation vectors for human gene therapy & plant genetic engineering (Yu et al., 2007)

Can be used in site-specific recombination or retrofitting the minichromosomes with additional foreign genes (Ow., 2007)

AC could be easily introduced or removed from a genotype by genetic crosses and would facilitate introgression of transgenes to different genetic backgrounds

It is possible that an important application of AC in plants will be combine them with haploid breeding (Ravi et al., 2010)

CONTD…

Summary Points…

Synthetic chromosomes in plants are likely to have more applications than in other taxa because of the ease with which they can be manipulated throughout their life cycle

Telomere-mediated chromosomal truncation works robustly in plants

Plant centromeres have an epigenetic component to their specification, which constrains the approaches to producing engineered minichromosomes

Site-specific recombination systems and zinc finger nucleases provide means to amend and grow synthetic plant chromosomes

It might be possible to develop a mini B chromosome-based genomic cloning system for capturing large chromosome fragments

Site-specific recombination systems & zinc finger nucleases are valuable tools for marker gene removal & gene targeting. Such technologies could be applied to AC

The mechanism involved in de novo telomere formation (DNTF) & capping is entirely unknown & are worthy for future study

Future Direction…Future Direction…