Lecture 4 - Methods of gene manipulation

Question 1

What is the action of agrobacterium upon plant wounding?

Answer 1

WT agrobacterium causes crown gall disease

1. If in the vicinity of wounded plant tissue this is recognised by the bacteria as the plant releases certain molecules
2. This activates an area on the tumour inducing plasmid in the bacteria containing the vir region, containing factors necessary to deliver parts of the plasmid to the cell. This is the t-DNA, and is boardered by recognition sequences for some virulence proteins
3. ssDNA is pooled off and tightly bound to virulence proteins so that it is protected
4. ss tDNA cannot reach the nucleus alone but the vir proteins contain nuclear location signals
5. ss tDNA moves to the nucleus and integrates within the chromosomes via illegitamate combination (not involving homolgous recombination)
6. DNA is integrated into the chromosome and converted into dsDNA

Question 2

What is the binary vector system?

Answer 2

Pair of plasmids consisting of a helper plasmid and a binary plasmid. Used together to produce genetically modified plants. Artificial vectors that have been created from the ti plasmid in agrobacterium. T-DNA is located on the binary vector, vir factors act in trans (on other plasmid) they are not necessary to be on the plasmid to deliver the T-DNA to plants. Contain a selectable marker so can select for integration. Genes between the left and right boarder sequences integrated into the genome.

In WT, the genes between border sequences are those that cause tumour growth, in BV system these have been removed. Ti plasmid v big so not good to work with in the lab but the BV system has been developed and is easier to work with.

Question 3

Give examples of binary vectors

Answer 3

pBIN19

• developed early on
• selectable marker near the right border
• kanamycin selectable marker

pGreen

• developed later
• selectable marker near left border
  
  • not every plant cell gets successful integration of tDNA into plant genome, region near the left boarder is transferred last, there may be a break point where only the right border is transferred and the gene of interest will be missing. Selectable marker near the left border is more beneficial
  • Kan selectable marker

Question 4

How is recovery of successful binary vector transformations acheived?

Answer 4

• regeneration of plants from transformed cells
  
  • plant tissue (select a part of the plant and incubate with agrobacterium. kill agrobacterium and grow tissue on a media with the appropriate mix of plant hormones (e.g. certain cytokines and auxin) plant tissue forms cells and as these are pluripotent plants can be regenerated from this tissue) -> callus -> shoot and roots
  • protoplasts -> callus -> shoot and roots
• floral dip (only arabidopsis + related plants e.g. chamdina but easier)
  
  • dip 4 week old (just started to flower) infloresence in agrobacterium culture
  • let plants set seed
  • plate seeds on a selective medium (those that grow have recieved the tDNA)

Question 5

What is the process of gene transfer by particle bombardment?

Answer 5

1. place plant material into a vacuum chamber
2. have gold particles covered in the gene of interest
3. sudden increase in pressure by rupturing disk
4. accelerates gold particles into the plant tissue and physically delivers the DNA
5. protoplasts and other types of plant tissue can be used

Question 6

What factors influence transgene expression?

Answer 6

Stages of gene expression

• transcription
• RNA processing
• mRNA turnover
• protein synthesis
• protein modification
• protein turnover

all these stages can be regulated allowing an optimisation of GM efficiency

Question 7

How is gene expression regulation?

Answer 7

1. Genetic mechanisms
   
   • ​​depend on nucleotide sequence
   • can be controlled precisely by construct design
   • e.g. promoter sequence, splice signals
2. Epigenetic mechanisms
   
   • Not as easy to deal with
   • some areas more easily silenced than others
   • do not depend on nucleotide sequence
   • cannot be controlled precisely by construct design
   • often depend on the site of transgene integration and locus organisation
   • e.g. chromatin structure, DNA methylation

Question 8

How can transcript initiation be controlled?

Answer 8

• By using appropriate promoters
• Promoters determine where the transcript starts and is controlled by the core/minimal promoter element
• Transcriptional start site is not just controlled at the nucleotide level
• Also distal elements which control the temporal/spatial expresssion (enhancers)

Question 9

What factors regulate transcription?

Answer 9

Constitutive promoters

• active in most plant cells most of the time
• useful for maximal accumulation of recombinant protein
• e.g. CamV 35s promoter, maize ubiquitin-1 promoter

Regulated promoters (cell-specific/stage-specific)

• activity is restricted to particular tissues or developmental stages
• e.g. maize zein (seed specific), rice sucrose synthase (phloem specific), histone (meristem specific)

Regulated promoters (inducible)

• active in response to external signals
• examples of native inducible promoters: rbsC promoter (light induced), PR-1a promoter (chemically induced)
• examples of heterologous promoters used in plants: E.coli lac and tet systems, mammalian hormone inducible systems. Advantage: no background level of exprssion in plants

Question 10

How can enhancer trap lines be used to identify tissue-specific expression?

Answer 10

Use gene encoding yeast TF which will activate yeast promoter that control the expression of a reporter gene.

2 step system, yeast TF only produces if upstream promoter is active, but the promoter is not active alone

Promoter is flanked by right and left border of T-DNA and in large screen is integrates at random points in the genome

If it integrates into the genome in areas near to control elements and enhancers that determine tissue specific expression the enhancers act together with the minimal promoter to activate the gene

TF is produced and binds to yeast promoter to activate gene, get reporter expression

Reporter: GFP

Need selectable marker for transgenic activites

Question 11

How can tissue specific expression of a gene of interest be achieved?

Answer 11

1. Use enhancer trap lines to identify tissue specific expression
2. Combine this us UAS-gene line - express a separate gene and cross the two transformed strains
3. Following selection, results in plants expression two T-DNAs, with one expressing an enhancer in the region of interest
4. If the other gene is activated by the yeast TF then can express any GOI in the same cells that the reporter gene was expressed in
5. e.g. if use a toxic gene then the gene expression location can be tightly controlled

Question 12

Outline how it was shown that dexamthasone does not adversely affect tobacco growth

Answer 12

1. Start with hormone inducible heterlogs
2. Young seedlings treatment with 20mM of DMT at fairly low concentrations can switch on a high level of expression of the gene
3. Can paint spots on leaf with DMT and get expression in particular location

Question 13

What is the difference between eukaryotic and prokaryotic protein translation
?

Answer 13

Eukaryotic

translation reinitation by ribosomes is unusual in eukaroyes

instead have a single coding region that is translated and then the ribsosome falls off even if there is another coding region following

sequence around the start codon is very important

Prokaryotic

have operons with a single mRNA and one coding region after another can be translated

have translation reinitiation by ribosomes

Question 14

what are the Kozak rules for protein synthesis?

Answer 14

Get good translational initiation if have AUG at +4 of start codon for eukaryotes and a A/G at position -3

In higher plants, as long as there is at least an A/G at +4 and a purine (A/G) at -3 then a sequence rich in A upstream of the A/G get much higher translational efficiency

Question 15

Why are the Kozak rules important in GE?

Answer 15

If want to express genes isolated from bacteria in plants. Often in bacteria have a C/T at the postions in higher plants where a purine would result in high translational efficiency. If find a purine at these positions then have ten fold less translation

Question 16

How were the effects of the 5’UTR on translational efficiency disovered?

Answer 16

• Looked at sequence 21 nucleotides upstream in arabidopsis using a reporter gene system (GFP) to study translational initiation efficiency.
• Looked at the relative mRNA levels but there was no substantial difference.
• Used betagalactosidase as different reporter, and studied numerous upstream genes from arabidopsis.
• Used western blot to demonstrate the presence of GFP and GFP fluoresence.
• Found huge differences depending on the sequence context - even if have a G/A at position +4, -3, can still have low level of translational initiation if the rest of the sequence is unfavourable.

Question 17

Why are upstream AUGs problematic?

Answer 17

Ribosomes start at the first AUG and often cannot restart at the downstream AUG. In higher plants and eukaryotes, dont have translational elongation

Poor translation if AUGs in 5’UTR are in a favourable context. Ribsomes will start at the first AUG, fall off an the region will b translated at low efificney.

Possible nonsense mediated decay if the upstream ORF is mmore than 100nk

Question 18

How can translation efficiency be enhanced by adding particular elements?

Answer 18

e.g. by translational enhancers of the tobacco mosiac virus

Used in vitro translation and then intoduced different transcripts into protoplasts and studied the reporter gene expression. (luciferase - easy to quantify)

Used luciferase coding region as basis and checked what elements are necessary for very efficient translation

Polyadenylation signal at the end of the coding region results in a dramatc increase compared to without PA signal.

PolyA site can be replaced by non-polyA 3’ site from TMV and get the same level of activity.

Used different cappings (eukaryotes have modification at the 5’ end of mRNA important for translational efficiency)

then used TMV delta sequence upstream of the luciferase coding region and even in the absense of polyA had a big expansion in expression (10 fold)

synergistic effect of delta (TMV 5’UTR) and TMV 3’ results in a further 5 fold increase

Question 19

Give an alternative to the enhancer trap system to identify positive control elements

Answer 19

• Large scale expression study in a microarray analysis or RNA sequencing
• Take tissue from plants and subject it to different conditions and see which genes are up/downregulated
• Identify the common elements upstream of upregulated genes
• Those genes that are expressed in a similar pattern would expect to have similar elements in promotor regions, identify conserved elements for genes that are reulated in certain conditions
• Combine elements and generate new specific promoters that can be used to trigger gene expression in certain conditions to optimise the expression of transgenes

Question 20

How can transcripiton termination be controlled?

Answer 20

• all transgenes must include a polyadenylation site
• mRNAs lacking a polyA site decay rapidly and absence may lead to read through onto other genes
• the CamV 35s and nos polyA termination sites used for transgene constructs

Question 21

How can intron splicing affect gene expression?

Answer 21

• in most cases GU at the 5’ site and a AG at the 3’ site
• plant introns are usually smaller than those of vertebrates (advantage: can often use whole genomic sequence in vector design)
• plants contain AU-rich sequences necessary for efficient splicing
• cryptic splice sites in heterologous genes result in very low expression levels
• use PCR to check

Question 22

How does mRNA decay affect gene expression?

Answer 22

• deadenylation-dependent decapping decay pathway (controlled by instability elements such as AUUUA in 3’UTR)
• nonsense mediatied decay pathway (controlled by premature stop codons) - defense system against incorrectly transcribed gene. If an exon containing a stop codon is followed by another exon it is degraded as a truncated protein may be detrimental
• RNA interference (against viral RNA’s but also control of indogenous gene expression)

Question 23

How does codon usage effect gene expression?

Answer 23

• degenerate genetic code
• codon usage varies
• certain codons in E.coli are used more often than in eukaryotes
• certain codons are not used often in plants
• associated enzymes that use these codons are less effective so should be avoided
• look at genes that have high levels of translation and the codon usage
• then manipulate transgene appropraitely to enhance translational efficiency

Question 24

Give an example of when a modification of codon use has been used successful to increase gene expression efficiency?

Answer 24

Engineering Cry1 genes for optimal Bt toxin production in plants

62/1743 bases modified and the effect observed on the bt toxin production

removed instability elements polyA signals internal to the sequence removed and codon usage optimised for plants

resulted in a dramatic increase in tomato and other transgenic plants in the level of toxin produced

up to 100 fold higher level of protein produced with fully modified construct

Question 25

How can efficient translation be attained in plants?

Answer 25

1. avoid very short 5’UTR around 80nk good
2. 5’UTR with low GC
3. avoid secondary structures in 5’UTR
4. No upstream AUGs
5. Favourable sequence context around AUG
6. Use translational enhancers (5’ UTR and introns)
7. Optimise codon usage adapted to plant species

Question 26

What are the features of position-dependent silencing? How can it be avoided?

Answer 26

• type of epigenetic regulation
• transgene integrates into repressed chromatin (heterochromatin)
• condensed chromatin structure and methylated DNA spreads into transgenic locus
• transgene is not transcribed

How to avoid

• insert transgene into a permissive site
• use boundary elements/MARs

Question 27

What are the features of homology dependent silencing and how can it be avoided?

Answer 27

Features

• affects multicopy transgenic loci (e.g using particle gun gets multiple integrations)
• multiple locations of genes may induce ectopic recombination
• inverted repeat structures may result in RNA self complmenting -> RNA silencing
• affects single transgenes homologous to endogenous genes
• endogenous genes can be silenced (cosupression)

Avoid

• single copy loci
• simple locus structure
• moderate expression levels

Question 28

What are the features of RNA silencing of single copy transgenes?

Answer 28

• Maximal transcription (if expressed through a strong promoter ) can saturate downstream processes such as polyadenylation
• any untailed mRNA may snap back resulting in the formation of a hair pin
• dsRNA induces RNA silencing
• transgenic plants may be fine for multiple generations but expression level may not be stable and expression can be switched off

Avoid

Introgression results in less silencing of transgenes which is likely due to the random insertion of genes

Question 29

How can transgene silencing be avoided?

Answer 29

• transgene locus should be protected from positional effects
• transgene should have a similar base composition to the host plant
• superfluous sequences, particularly on the plasmid backbone, should be avoided
• the transgene copy number should be kept as low as possible
• the locus structure should be simple, preferably a single intact copy of the transgene
• inverted repeat structures should be avoided
• maximised transcription may be counterproductive

Question 30

What are the three methods of gene targeting?

Answer 30

Reverse genetics

• Want to manipulate a gene sequence where the sequence is known
• Identify plant lines with a mutation in a specific gene
  
  • Screen for point mutations: TILLING (random introduction of point mutations and create library of mutant plants. Use heteroduplex mapping to identify point mutations in the GOI using PCR approaches
  • Screen for isolation mutants
  • Screen for deletion mutants

Directed mutagenesis

• Targeted generation of small mutations in a pre-determined gene (easy to generate mutations, through large scale T-DNA transformation which is incorperated into the genome and if it inserts into the coding region of the genome get K/O which can be identified by PCR approaches using primers that are complementary to the T-DNA)

Gene replacement

• Insertion of a transgene in precise location (Generate deletion mutants then use PCR to identify deletions)

All expensive and time consuming

Question 31

What is the process of gene targeting by zinc finger nucleases?

Answer 31

1. Use site specific nucleases (engineered zinc finger nucleases) to generate double stranded breaks at specific places in the genome
2. dsbreaks lead to NHEJ for repair - involves a number of proteins to degrade the ends, process the ‘sticky ends’ and put them back together
3. Results in mutations (INDEL) and if there is an INDEL in a coding region of a gene get a K/O
4. Can introduce a repair template to go across the break site and then if this DNA has sequence homology left and right of the break site the break can be repaired by homologous recombination
5. DNA synthesis will continue using the intact template (sstemplate) leading to gene modification

Useful in crop cultivars if have a SNP that contributes to crop performance

Question 32

What are the features of zinc finger nucleases?

Answer 32

protein domains that bind zinc/iron found in certain transcription factors

Can be optimised by a selection system in yeast to bind to certain trinucleotide sequences

Can increase specificity by combining multiple zinc finger domains fused to endonuclease Fok1, increased number of ZF domains = higher specificity

Allows the generation of site specific breaks

Question 33

How was gene replacement achieved in maize through ZF nucleases?

Answer 33

1. Use of the ZF nucleases to cut a specific locus in two
2. Integration of a transgene by HR
3. Gene of antnutritional properties (phytate synthesis) disrupted by the integration of herbicide tolerance gene

Question 34

How was it shown that ZF nucleases discriminate between highly similar genes?

Answer 34

1. Targeted two sites in exon 2 Zea mays IPK1 gene with two different engineered nucleases. Target gene had close homology elsewhere in the genome with a single nucleotide difference
2. Observed the activity of nucleases against the correct target and showed very high sequence specificity

Question 35

How was it defined that ZF nuclease GE results in mendelian inheritance?

Answer 35

1. Introduced ds breaks by ZF nucleases and analysed a large number of sequences
2. Demonstrated that mostly acheived deletions and some insertions, due to NH recombination
3. when the repair template carrying information for basa resistance was integrated into the genome found had the inclusion of both alleles in some lines. Some were a variation. (PCR)
4. Plants were selfed and checked to see whether the insert was passed onto the offspring -> backcross, observed expected mendialian pattern

Question 36

What are the disadvantages of using ZF nucleases?

Answer 36

• complicated
• expensive
• not practical for every lab

Question 37

What can be used as an alternative to ZN nucleases?

Answer 37

TALE (transcription activator-like effectors) nucleases

TALEN

Question 38

What are TALEs?

Answer 38

Genes isolated from plant pathogenic bacteria with a repeat structure (identical amino acids repeated)

Repeat variable di-residues determine the specificity for each of the repeat domains and bind to specific bases

By combining the modules of repeats in a protein, the proteins bind to specific DNA sequences

Question 39

Compare ZFNs to TALENs

Answer 39

Both combine specific domains with nucleases -> site specific nucleases

TALEN protein bigger than ZFN so harder to handle

TALENs can be designed so don’t have to go through yeast selection system, can be directly generated and used in construct design

Question 40

Why is wheat breeding tricky?

Answer 40

Bread wheat is hexaploid (17GB)

Human genome 3GB

Corn 2.3 GB

Soybean 1.2 GB

Rice 0.45 GB

3 genomes (A, B, C) make it difficult to do mutation breeding, if introduce a mutation there may often be a corresponding gene in another of the genomes

Question 41

Why are plants with one genome easier to manipulate than those with more?

Answer 41

Bread wheat (3 genomes): one genome can be made homozygous by selfing but there will be no crossover with other chromosomes therefore difficult to get a triple mutant and K/O in the desired gene

Question 42

How can powdery mildrew resistance be acheived in bread wheat?

Answer 42

1. MLO gene K/O can achieve powdery mildew resistance in crop plants
2. Tried to K/O MLO in hexaploid wheat using TALEN system. Used conserved region MLO in exon 2, which is conserved between the 3 genomes.
3. Targeted regions such that the nuclease would cut at the region with restriction endonuclase site and introduce INDELs to remove the restriction endonuclease site
4. Test whether successful by PCR then cut with enzyme and if the region is not cut then the region has been altered
5. simultaenous K/O of the three homeoalleles of the MLO locus in wheat acheived by identifying individuals with an uncut band in all three genomes
6. plants selfed and the K/O in all three genomes resulted in dramatic success - if one/two genomes affected plants were susceptible to powdery mildew but get complete resistance in triple mutant, hyphae did not enter the plant tissue
7. after segregation of the transgene, these plants can be made free of foreign DNA

Question 43

How was targeted insertion of GFP in the MLO locus acheived via NHEJ?

Answer 43

1. A template with GFP coding region flanked by cutting sites was delived into a cell where a site specific nuclease would cut at the MLO gene
2. This would generate sticky ends and result in integration of the GFP coding region - combination of techniques, integration of DNA through site specific integration and NHEJ (useful for plants as HR inefficient - most dsbreaks in plants are repaired by NHEJ followed by the HR repair system)

Question 44

What is the CRISPR/Cas adaptive immune system in bacteria?

Answer 44

1. When some bacterial strains are attacked by bacteriophages, they take a part of the bacteriophage genome and integrate it into their own genome.
2. This is then used as part of the immune memory of the bacteria
3. If the bacteria is attacked again, a transcript of the DNA phage genomic region is made
4. Transcript folds back to produce loop structure which is recognised by CAS endonuclease. This binds to small RNAs,which then bind by complimentary base pairing to a region in the phage genome
5. The CAS endonuclease cuts phage DNA so the bacteriophage cannot multiply

Question 45

How is the CRISPR/cas9 system used in genome editing?

Answer 45

• Guide RNA - target complementary system - 20 nucleotide sequence which can be designed at will - has a sequence that is common to all guide RNAs, loop recognised by the nuclease. Nuclease binds to the loop and the guide sequence binds to the specific target in the genome. Nuclease then adds a ds break (better then TALEN/ZFN)

Question 46

How can the CRISPR/cas9 system be modified?

Answer 46

• can be modified - centre that cuts DNA can be removed so that only a single strand is cut (endonucleosome -> nickase)
• advantage: can increase selectivity as need two guide RNAs which cut in adjacent sections of DNA
• disadvantages: not any sequence can be targetted. need to adhear to certain motifs at the endof the target region (NGG section)
• if inactivate nuclease activity completely cas9 can be used as a vehicle to direct any other protein attached to cas9 to a region in the genome (e.g. a transcriptional activator/repressor) and then used to manipulate gene expression

Question 47

How did Zhang use the CRISPR/cas9 system in rice?

Answer 47

Used to produce specific and homozygous targeted gene editing in rice in one generation

Normally have one genome editied but not the second, showed could manipulate a section of genes in rice using different guide RNA