Synthetic chromosome seminar

May 3, 2023 Department of Genetics and Plant Breeding 1

WELCOME


Poornima.R.N.PALB 4214

Senior M.Sc

May 3, 2023 3

Flow Of Seminar

Department of Genetics and Plant Breeding

Introduction. Need of Synthetic Chromosomes. Requirements for Synthetic

chromosomes. Methods to develop Synthetic

chromosomes Advantages and limitations. Application of Synthetic

chromosome technology in crops- Case studies.

Future Aspects. Conclusion.


Food In Fifty Years…??


Why make Synthetic chromosomes…???


Synthetic Chromosomes

Knowledge

Products

May 3, 2023 Department of Genetics and Plant Breeding

Need of Synthetic/Artificial chromosomes

Direct method

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• Gene stacking- difficult• Transgene position

effects

First Generation Genetic Engineering

Second Generation Genetic Engineering

Synthetic Chromosomes

Delivery of large DNA sequences

Complete metabolic pathways

Genetic changes 100-kb to megabases (Mb)

Need.....???

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Synthetic chromosomes are chromosome based non-integrating vector system that is transmissible and suitable for transfer of large genes, gene complexes and/or multiple gene together with regulatory element for safe, controlled and persistent gene expression

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03/05/2023 DEPARTMENT OF PLANT BIOTECHNOLOGY 10

Demonstrated the successful use of telomere truncation in maize plants to produce minichromosomes (2006)

created a synthetic chromosome from yeast paving the way for a new

field of study(2014)

Pillars of this new technology

James A Birchler

J. Craig Venter


“This has been sought for a long time in the plant world, and it should open a number of new avenues. If we can do this in plants, a number of advances could be done in agriculture that would not otherwise be possible, from improved crops to inexpensive pharmaceutical production to other applications in biotechnology”

James BirchlerMU College of Arts and Science

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Requirements for Synthetic Plant Chromosome

Basic requirements for Synthetic Plant Chromosome

1.Centromere

2. Telomere

3. Sufficient Chromatin

4. Selectable Marker transgene

5.Site specific recombination system

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Centromere Components In Plants

Centromere – Requisite component of SC

Contains tandem repeated sequences of various sizes, ~1 to 3 Mb.

CENH3,the Histone H3 varient of plants typical of active centromeres, associates with this repeat region. Retrotransposons (Maize-CRM elements)

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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 (Burr et al., 1992) Functions: 1. Maintaining the structural integrity

2.Ensure complete replication of extreme ends of chromosome

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Methods To Develop Synthetic Chromosome

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Bottom up method

• de novo assembly of cloned chromosomal components, such as

centromeric and telomeric sequences, a selective marker gene and genomic DNA that contains a replication origin.

This method is well established inYeast (Murray and Szostak, 1983) Mammalian cells (Harrington et al.,1997)

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Reasons for failure

• Construction yeast artificial chromosome is used as a model for bottom-up method.

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

e.g. Centromeric repeats of barley (Hordeum vulgare) have been shown to be neither necessary nor sufficient to establish a functional centromeric activity (Nasuda et al., 2005) .


Epigenetic Aspects Of Centromere Specification

• Mechanism by which centromeres are established, maintained,

and function remain a mystery.

• A chromosome was found that did not contain detectable

centromere repeats but till organized a kinetochore at a specific

site that was associated with centromere activity in barley

(Nasuda et al.,2005).

• Substitution of the histone H3 by CENH3(CENP-A) in

centromeric nucleosomes is crucial for kinetochore formation.

18Department of Genetics and Plant BreedingMay 3, 2023

So what is the solution…???



Top down methodBased on chromosome fragmentation or truncation.

This method utilizes the insertion of telomere sequences into existing chromosomes.

This sequence signals for new telomeres which causes the truncation of the chromosome.

insertion of new genes for desired traits.

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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)

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Adding of genes to a engineered minichromosome

This approach of telomere mediated chromosome truncation was first shown by Farr et al.,1991 i.e. introduction of cloned telomeric repeats into cultivated cells may truncate the distal portions of chromosomes by the formation of new telomeres at integration sites in mammalian cell lines.


Type of truncation

Description Pros Cons

Truncation of Bchromosomes

In plant species that harbour Bs, truncation of B chromosome arms can be selected.

Loss of chromosome segments from Bs does not affect the viability of the plant.

Bs are not present in all plant species. This approach is applicable only to those speciesthat have or will accept Bs.

Truncation of Achromosomes in

tetraploid background

Truncation of Achromosome can be rescuedin a tetraploid background.

Tetraploid plants can tolerate truncation of chromosome arms. This approach will work readily in natural polyploids.

Tetraploids would need to be generated in diploid species.

(Gaeta et al.,2012)24Department of Genetics and Plant BreedingMay 3, 2023


A chromosome truncation B chromosome truncation

Particle bombardment

Truncating plasmid

Telomere truncation of different types of chromosomes

(a) A chromosome

(b) B chromosome

(c) Telocentric chromosome



Minichromosomes

• extremely small version of a chromosome.• By minimizing the amount of unnecessary genetic information

on the chromosome and including the basic components necessary for replication,we can construct this chromosomal plotform,so that we can insert new genes.


B Chromosome Based MinichromosomesB 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



Generalized scheme for the production of engineered minichromosomes in maize

J. A. Birchler et al.,2010


Maintenance Of mini B chromosomes in the population


Advantages and Limitations

• Fecilitates in understanding fundamental questions about chromosomal structure and function.

• Synthetic chromosomes in plants are likely to have more applications than in other taxa.

• This approach for construction of engineered chromosomes can be easily extended to other plant species

• allows for the stacking of genes side-by-side on the same chromosome thus reducing likelihood of segregation of novel traits.

Advantages


• Functional genomic studies could use minichromosomes as a platform for adding specific genes

• Next generation vectors for human gene therapy & plant genetic engineering

• stable expression & maintenance of multiple transgenes in one genome.

• Mass production of foreign proteins, pharmaceuticals, or useful metabolites.


Limitations

• Small chromosomes do not always pair homologously in meiotic prophase.

• low meiotic transmission rate.

• pollen abortion.

• Regeneration of plants is difficult.


Case Studies



• In this report, they demonstrated that 2.6 kb of Arabidopsis telomeric repeats were efficient for telomere-mediated chromosomal truncation in maize.

• They also showed that internally integrated telomeric sequences are stable in the genome.



Constructs Transgenic plants

Transgenic loci

pWY76 93 123(57 at distal loci)

pWY86 83 108 (61 at distal loci)

pWY96 44 58 (11 at distal loci)


Cytological detection of chromosomal truncations

pWY86 pWY76

pWY86 pWY86

B77

T87

B37B44


Restriction mapping of the positions of transgenes by a Southern blot.


Summary• 2.6-kb direct repeat of Arabidopsis telomeric sequence

was used in two constructs, pWY76 and pWY86, to test the ability of telomeric sequences to cause chromosomal truncations.

• Direct evidence for chromosomal truncation came from the results of FISH karyotyping, which revealed broken chromosomes with transgene signals at the ends.

• This technology will be useful for chromosomal engineering in maize as well as other plant species.

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EXPERIMENTAL PROCEDURES

Plant material: Embryogenic calli derived from Immature embryos of rice telotrisomic line with Telo-12L derived from indica rice cultivar Zhongxian 3037.

Callus induction: • Immature embryos were cultured on N6D2 medium at 28 ͦC in

the dark for callus induction.• Embryogenic calli were subcultured every 2–3 weeks and used

for transformation by bombardment.


Plasmid construction


These three plasmids are mixed together in the ratio of 5:1:1

Gene transformation:• 1.5 mg 0.6 μM gold particles were coated with 2 μg plasmid mixture

and bombarded to rice calli with a PDS 1000/He particle delivery system.

• After 16 hours,the calli were transferred onto N6D2 medium supplemented with 25 mg/L hygromycin and cultured at 28 ͦC in the dark for 2 weeks.

• and then selected by subculture on N6D2 supplemented with 50 mg/L hygromycin every 3 weeks until the resistant calli emerged.

• The resistant clones were maintained on N6D2 medium supplemented with 25 mg/L hygromycin at 28 ͦC by subculture every 3 weeks.

Chromosome preparation from callus• Fast growing calli on selection medium after 7 days subculture were

selected to check chromosomal truncation and mini-chromosome formation in transgenic rice using FISH.


(b)Transgenic clone with one distal signal

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(a)Transgenic clone with one internal signal

Fluorescence in situ hybridization(FISH) detection of transgenes

Rice transformation and transgene distributions

No. ofbombardedcalli

No. of resistance clones

No. ofTransgenic eventschecked

No. ofTransgene signals

No ofinternaltransgenes

No. ofdistaltransgenes

Ratio (%)of distaltransgenes

Control (a) 519 44 19 32 31 1 3.1

4–6-month-old calli (b)

4512 536 122 178 121 57 32.0

18-month-old calli (c)

854 111 94 138 84 54 39.1

Total no. (b + c)

5366 647 216 316 205 111 35.1

03/05/2023 48

The control was bombarded with only pCAMBIA1301


(a–c) Mini-chromosomes with one transgene signal: (a) Clone 1008-100(b) Clone 1004-111(c) Clone 1004-015.(d) Clone 1004-011, mini-chromosome with transgene signals at both ends of the chromosome.

Rice mini-chromosomes from telomere truncation.


Clone 1004-111

• One chromosome 12 and one Telo-

12L are shown in this clone.

• The mini-chromosome

(arrowhead) is probably originated

from chromosome 12 truncation

Origin of mini-chromosomes


Clone 1008-100

• Three chromosomes 12L are identified

• Original Telo-12L was not found.

• The missing 12L is probably truncated to

produce the mini-chromosome

(arrowhead).

• Chromosome 12 is probably duplicated

to produce an extra chromosomes 12.


Distal transgene in control without

telomere sequence

Distal transgene with telomere

sequence Maize

chromosomal truncation

19.0 %

46.3-56.5 %

Rice chromosomal truncation

3.1 %

35.1 %

These results were compared with previous report of maize chromosomal truncations by Yu et al.,2006

Summary

• Telotrisomic rice line was used for mini-chromosome construction.

• minichromosomes were recovered from transgenic clones by monitoring hygromycin-resistant calli with FISH.

• All these mini-chromosomes were maintained stably in cell cultures for over 2 years.

• The construction of mini-chromosomes in rice will provide a platform for future artificial chromosome-based genetic engineering of this plant for multiple gene expressions.



Future Direction

• develop a mini B chromosome-based genomic cloning system.

• combination with haploid breeding.

• Genome editing techniques. • Deliver genes that benefit the

agricultural, nutritional, energy and pharmaceuticals sectors.

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Conclusion


Thank You


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Synthetic chromosome seminar