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3s2: Plant biotechnology > Week 3 > Flashcards

Week 3 Flashcards

1
Q

What is an overview of Brassica oleracea?

A

Wildtype selected into 9 widely eaten crops:
Brussel sprouts
Romanesco
Broccoli
Kale
Cabbage
Cauliflower
Kohlrabi
Red cabbage

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2
Q

What is transgenic?

A

If there is no existing genetic variation for a trait within a species / or closely related species, genetic sequence can be taken from another species
Transgenic = different

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3
Q

How much money does it cost to bring GM crop to market?

A

It takes ~$115M to bring a new GM crop to the market

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4
Q

What costs/benefits for GM crops?

A

Farmer benefit – increased profitability
Supply chain / producer demand/ acceptance
Customer demand/ acceptance
Regulatory barriers
Trade considerations

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5
Q

How much has global area of biotech crops between 1996 to 2019?

A

1996 - 1.7 million hectares
2019 - 190.4 million hectares - 10% of total global agricultural land area

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6
Q

What is the big 5 countries of GM crops?

A

USA - 37% largest producer
Brazil - 28% largest developing producer
Argentia - 13%
Canada - 7%
India - 6% more than 6 million farmers planted 11.9 million hectares in cotton in 2019

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7
Q

What are approved GM crops that will soon reach the market?

A

Golden Rice
Drought tolerant wheat – Argentina 2020, Brazil 2023
Insect resistant cowpea - Nigeria 2019, Ghana 2022

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8
Q

What 4 crops accound for 99% of all GM grown?

A

Soybean - 50%
Corn - 31%
Cotton - 13%
Oil seed rape - 5%

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9
Q

What makes up most of the remaining 1% of GM grown?

A

Alfalfa - 0.5%
Sugar bees - 0.3%
Sugarcane - 0.3%

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10
Q

What is an overview of crops and the market share of GM crops?

A

Cotton - 76%
Soybean - 78%
Maize - 30%
Canola - 29%

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11
Q

What is an overview of the labelling of GM crops in the EU?

A

No labelling requirements for nonfood use or feed
Food Labelling required ie Resse’s pieces
UK still following EU GMO directive 2001/18

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12
Q

What is the overview of the costs of weeds?

A

Weeds = major cause of yield loss
Soybean = losses of up to 50 % (est. loss $16Bn)

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12
Q

What are the GM traits that dominate the market?

A

Herbicide Tolerance (HT) - 47%
Insect Resistance (IR) - 12%
Stacked Traits HT/IR/other - 41%

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13
Q

What is an overview of herbicide tolerance?

A

Resistant to broad spectrum herbicides e.g. glyphosate – commonly used by conventional farmers and gardeners

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14
Q

What is an overview of glyphosate?

A

Glyphosate works by inhibiting an enzyme called EPSPS in the plants necessary for the production of the aromatic amino acids, auxin, phytoalexins, folic acid, lignin and many other secondary products

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15
Q

What is an overview of the two forms of EPSPS?

A

There are two forms of EPSPS in nature, EPSPS I, which is found in plants, fungi, and most bacteria, and is sensitive to glyphosate, and EPSP II, which is found in glyphosate resistant bacteria and is not inhibited by glyphosate
It is the gene for an EPSPS II that has been used to genetically engineer resistance in crops

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16
Q

What is an overview of the use of glyphosate?

A

EPSPS not found in animals, therefore the herbicide is not toxic to humans

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17
Q

What is an example of microbial insecticides?

A

Use of the bacterial spray Bacillus thuringiensis (Bt)
B.t produces a protein which is lethal to the target pests, but harmless to non-target insects and mammals
Used by conventional and organic farmers

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18
Q

What is an overview of B.t insect resistance cotton?

A

Bt cotton now helps to avoid several million cases of pesticide poisoning in India every year
GM introduced in 2002 and by 2014 India became the No1 Producer of cotton
1960 - 5 million bales
2002 - 15 million bales
2020 - 35 million bales (20 million with introduction)

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19
Q

What is the overview of Bt Brinjal?

A

Bangladesh South Asia’s first approved GM food crop 2013
In 2014 it was grown by 120 farmers, by 2021 over 60, 000 farmers - now represents >50%
Reduced pesticide inputs (80%) and spraying labour costs
The fruit is highly desirable because it is undamaged by pests and can be sold in markets labelled as free from insecticide
Highly profitability for smallholder farmers

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20
Q

How have GM crops impact farmer outputs and inputs?

A

Yield - +22%
Pesticide quantity - -37%
Pesitcide - -39%
Total production cost - +3%
Farmer profit - +68%

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21
Q

What is an overview of GM crop for drought tolerance?

A

DT corn approved in US - 2013
US 2012 drought estimated at $17Bn in farmer crop losses

DT/IR sugarcane – Indonesia - Approved

Drought and salt tolerant soybean - Argentina 2019

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22
Q

What is an overview of virus resistance papaya?

A

Papaya on hawaii no natural protection form Papaya ringspot virus
Papaya ringspot virus nearly destroyed the
Hawaiian papaya industry
Alternative control would be to destroy infected trees

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23
Q

What are examples of GM products aimed at reducing food waste?

A

Non-browing apples - RNAi to silence Polyphenol oxidase (PPO) gene
2015/2016 US approved 3 GM apple varieties
(Granny smith, Golden Delicious and Fuji)
As of 2020 there were 1,350 acres (550 ha) of Artic apple orchards in Washington state = 7.7MKg harvested in 2021
Reduced browning potatoes

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24
Q

What are GM potatoes?

A

3 genes from a wild relative: blight resistance, A genetic approach to reduce the need for chemical inputs
Improved tuber quality / cold storage

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25
Q

What is an overview of golden rice?

A

Boost Vitamin A production
Vitamin A deficiency responsible for 1-2 million deaths and 500, 000 cases of irreversible blindness/ year
A serving of Golden Rice contains half the beta-carotene children need daily

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26
Q

What is an overview of the approval of golden rice?

A

Australia 2017 (Food use)
New Zealand 2017 (Food)
Canada 2018 (Food)
US 2018 (Food/Feed use)
Philippines 2019 (Food/Feed use)
Philippines 2021 cultivation

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27
Q

What is an overview of the distrubution of golden rice?

A

October 2022, 67 tons harvested from 17 fields.
To be distributed to households with pregnant women, breastfeeding mothers or preschool children who are at risk of diseases caused by vitamin A deficiency

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28
Q

What is an overview of GM crops for nutritional quality?

A

Pink Pineapples - higher in anthocyanins and therefore antioxidants added health benefits
Grown in Costa Rica by Del Monte - US approval, 2016 Canada approved 2021

Purple tomatoes - Gained regulatory approval in the US in 2023 aimed at ‘home growers’ market – seed went on sale this month. $20 for 10 seed

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29
Q

What is an overview of EU countries of GM grown food?

A

Romania approx 150,000 ha in 2006 but heavily reduced in 2007 when joined EU
Spain consistant 50,000 ha 2006 to 140,000 in 2013

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30
Q

What is an overview of EU vs US cultivation and import of GM crops?

A

EU - 2 events cultivated and 140 events imports
US - 185 events cultivated and 218 events imports
Event = the gene and where it inserts in the genome

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31
Q

What politices causes EU to be slow with accepting GM crops?

A

European Food Safety Authority (EFSA) - Evaluates human and environmental risks of GM foods safe products vote by Members states - majority vote for or against needed goes to European Commission to decide
Often caught in a feedback loop

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32
Q

What is an overview of the EU cultivation proposal?

A

The aim was to help overcome the bottleneck in the EU approvals system.
Allows EU member states to restrict or ban the cultivation of GMOs on their own territory, even if allowed at EU level
GM opt-out - Germany, france and Italy
No opt-out - UK, Spain and Sweden

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33
Q

What is the consequence of the EU cultivation proposal?

A

Bt Maize approved in 1998, has a 20+ years safe history of use, yet its renewal remains pending – but is approved for import

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34
Q

What is an overview of gene editing and the politics?

A
  1. Delete/edit existing DNA
    In England this is referred to as a Precision Bred Organism (PBO) New legislation came into force March 2023
  2. Introduce/replace DNA at a precise location
    Regulated as GMO if the end point couldn’t be achieved by traditional breeding approaches i.e. transgenics

Scope for cisgenics under new legislation

35
Q

What is an overview of gene editing regulation?

A

Choosing to regulate in line with traditional breeding with some additional oversight/ notifications required - USA, India, Japan, UK
or
Continuing to regulate under GMO regulations - South Africa and New Zealand

36
Q

What is an overview of UK: New Genetic Technologies (Precision Breeding) Act?

A

Required changes to primary legislation: to amend the definitions of a GMO to exclude organisms that have genetic changes that could have been achieved through traditional breeding methods

37
Q

What is a history of the UK: New Genetic Technologies (Precision Breeding) Act?

A

2022 - Defra proposes changes to legislation
Ahead of completion in April 2022 – rules relaxed on
experimental field trials
2nd legislative changes are now needed to implement the Act, these are expected end 2024 for plants and 2025 for animals

38
Q

What is an overview of EU of NGTs?

A

Feb 2024 MEPs majority vote in favour of NGTs

39
Q

What is an overview of GM growing experiments?

A

JIC conducted ‘field trial’ gene edited tomato - edited to have higher levels of provitamin D3
UV (outside) light converts the provitamin D3
to Vitamin D3 (not seen under glasshouse conditions)

40
Q

What is an overview of gene edited wheat?

A

2023: Gene edited wheat – altered grain size
2023: On-going High Iron GM Wheat
Needs to be properly protected against to prevent spread of seeds ie cut seed producing organs and not grow things for several years to prevent accidental mixing in

41
Q

What are the benefits of GE to farmers?

A

Reduced inputs
Pest resistance
Disease resistance - Rust resistance (BCAT1)
Adaption to climate change - Drought tolerant Soybean
Cowpea edited for Mechanized Harvesting

42
Q

What is an overview of benefits to people for GE food?

A

Increased yields
Reducing yield loss/ food waste
Healthier foods - High GABA food, nutrient dense salads and healtier oil soybeans

43
Q

What is an overview of the different strategies for plant breeding?

A

Conventional breeding - non-targeted
Genetic modification - contains ‘foreign’ DNA
Gene editing - eg CRISPR does not contain ‘foreign’ DNA

44
Q

What was plant breeding resulted in?

A

Plant breeding selecting for genetic variations/mutations to meet human needs
Mutations have been selected for over the years to give us these different forms

45
Q

How can gene editing circumvent the downsides of plant breeding?

A

Variations/mutations by Gene Editing are indistinguishable from natural mutations

46
Q

How can mutation frequency be increased?

A

Increasing the mutation frequency by chemicals or irradiation

47
Q

Why is mutation generation useful for plant breeding?

A

Exposure to radiation induces changes in DNA, boosting mutation rates by 1,000 to 1 million-fold, which enables breeding plants more effectively and producing more crop variations in shorter time.

48
Q

What is an overview of mutation changes in the 3 biotechnologies?

A

Mutation breeding - Mutation changes - >500,000bp
Transgenic - 1 or more genes
Gene editing - 1 or more genes

49
Q

What is an overview of type of mutation in the 3 biotechnologies?

A

Mutation breeding - Random
Transgenic - Targeted
Gene editing - Targeted

50
Q

What is an overview of whether change could occur naturally in the 3 biotechnologies?

A

Mutation breeding - Yes
Transgenic - No
Gene editing - Yes

51
Q

What is an overview of whether foreign DNA is involved in the 3 biotechnologies?

A

Mutation breeding - No
Transgenic - Yes
Gene editing - No but possible

52
Q

What is an overview of transgenic step requried in the 3 biotechnologies?

A

Mutation breeding - No
Transgenic - Yes
Gene editing - Yes but not with new methods

53
Q

What are examples of genome editing technologies?

A

Zinc fingers
Talens
CRISPR

54
Q

What are the outcomes for gene editing technologies?

A

Double stranded break either NHEJ or HR
NHEJ knockout by insertions / deletions caused by error prone DNA repair
HR knockin by gene replacement / insertion

55
Q

What is an overview of CRISPR?

A

An adaptive bacterial defence strategy

56
Q

What is an overview of the history of CRISPR?

A

1993 – Discovery of CRISPR and its function (Mojica) * 2005 – Discovery of Cas9 and PAM (protospacer adjacent motif (Bolotin)
2010 – Cas9 cleaves target DNA causing a double strand break 3 nucleotides from the PAM (Moineau)
2012 – Cas9 can be reprogrammed to target a chosen site (Charpentier and Doudna)
2013 – CRISPR-Cas9 harnessed for genome editing in eukaryotic cells (Zhang)

57
Q

What is an overview of the history of CRISPR/Cas9?

A

The CRISPR/Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats)
Cas9, an RNA-guided DNA endonuclease
Cas9 is targeted to specific genomic sequences by a separately encoded guide RNA
Creates a double stranded break in the genomic DNA.
DNA repair enzymes, mistakenly cause insertions or deletion (Indels)
Indels cause frameshifts in the amino acid sequences and early stop codons

58
Q

What is the target of CRISPR?

A

The target - 20bp followed by the PAM motif (NGG)

59
Q

What is a simplefield overview of CRISPR?

A

New tool in our tool box
Cas9 protein + Guide RNA
Cuts DNA at precise location
Creates mutation at specific place

60
Q

What is the main components of CRISPR?

A

A guide RNA which searches the genome looking for the corresponding DNA sequence
The Cas protein that acts as a pair of molecular scissors which are able to cut the DNA (edit) at a precise location

61
Q

What is needed for desinging sgRNAs?

A

Target sequences and sgRNAs both conform to a GN20-nucleotide long sequence
However only targets contain a 3’-NGG PAM (protospacer adjacent motif)
The sgRNA protospacer must not include the PAM
The sgRNAs are cloned downstream of a TaU6 or TaU3 promoter and upstream of a ubiquitous 3’ hairpin loop (annotation) referred to as the scaffold.
The first G in GN20 is necessary for U6 transcription start (An A for U3). If a G is not present, it is possible to add an additional G

62
Q

What is important when desinging sgRNAs?

A

The guide RNA’s that you choose do not contain the restriction enzyme sites that you are going to be using during cloning
The termination signal for Pol III promoters such as the U6 or U3 is a line of 5 or more T’s

63
Q

What are common golden enzymes?

A

BsaI, BpiI, and in some systems BsmBI

64
Q

What is the relationship between tolerance of mismacthes tolerance?

A

Futher from the PAM sequence the greater the mismatches tolerated

65
Q

What are the features of domesticated wheat?

A

Three copies of every gene
~97% sequence similarity
Similar expression patterns (for the most part)
Huge plasticity and dynamic genome
Buffering effects of the genome

66
Q

What is needed when designing sgRNA for wheat?

A

When designing guides to target a single gene copy in wheat the ~97% sequence similarity between gene copies can on occasion be difficult.
When designing guides to target all three copies of a gene the sequence similarity between gene copies can be advantageous

67
Q

What needs to be considered when screening for CRISPR edits?

A

Primers should be designed that flank the target area.
Subgenome specific primers are require for each target gene copy
PCR amplicons are directly Sanger sequence
Alternatively Next Generation Sequencing (NGS)

68
Q

What is an overview of screening for CRISPR edits NGS Illumina?

A

Primers are designed that flank the target areas of all 3 homeologs
Multiplex PCR performed
Small amplicons of 100-180 bp * Amplicons should not differ in size +/- 10 bp
Primers of the separate amplicons should NOT overlap
If both guides work simultaneously leading to a large deletion, the multiplex PCR would result in an amplicon from FP1 and RP2.

69
Q

What is a golden gate assembly?

A

Golden Gate Cloning or Golden Gate assembly is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes and T4 DNA ligase

70
Q

What is an overview of golden gate assembly?

A

Uses type IIS restriction enzyme such as BsaI which miss a base before cutting and leave a sticky end
PCR components adding BsaI sites
Adding promoter, coding sequence and terminator
Add components, BsaI, Ligase and vector to a single tube
Use blue and white screening such as with LacZ to determine if properly functioned

71
Q

What is an overview of stacking with golden gate assembly?

A

By using vectors with addition Type IIS restriction enzymes such as Bpil
It is possible to build up very complex molecules quickly and efficiently
Useful for making gene stacking vectors
Or transcription activator-like effectors nuclease (TALEN) vectors used in genome editing experiments

72
Q

What is an overview of golden gate modular cloning system?

A

Level 0 the promoters, coding regions and terminators exist as single components
Level 1 the promoter, coding regions and terminators are joined to a level 1 acceptor by Bsal
Level 2 the level 1 acceptors are dropped into the level 2 vector by Bpil

73
Q

What is the advanatages of Golden gate MoClo system?

A

Standardised overhangs therefore components can be shared globally eg send promoter and will fit system

74
Q

What is an disadvantage of the CDS1 gene with gloden gate MoClo system?

A

AATG overhang is apart of the coding region

75
Q

What is an overview of the method for golden gate MoClo system?

A

Oligonucleotides ordered by sgRNA overhangs
The Level 0 complementary oligonucleotides are dropped into Level 1 TaU6 acceptors which contains the LacZ and scaffold
Multiple of the level acceptors are ligated into L2 acceptor with a linker region to go between the scaffold and right border region

76
Q

What is the final step for wheat transformation?

A

Once constructs are prepared and sequenced they
are transformed into wheat via Agrobacteriummediated transformation

77
Q

What are the pros and cons of the screening method sanger sequencing?

A

Pros: Large amplicon, relatively cheap, quick, and easy analysis.
Cons: Primers difficult to design/optimise for multiple targets/polyploids

78
Q

What are the pros and cons of the screening method NGS illumina?

A

Pros: Single primer pairs can amplify homeologs, accurate and highly detailed data. Quantifiable editing within a sample.
Cons: Small amplicons make identifying large multi-guide deletions difficult. Access and understanding of high powered computing required

79
Q

What is an easy way to screen for large deletions?

A

Through PCR, should show noticable difference in band size

80
Q

How do you know with sanger sequencing if a cut has been made?

A

Sudden drop in easy to read sequencing
Post cut there will be confusion in what base identity

81
Q

How do you know with NGS if a cut has been made?

A

Most knockouts in wheat require the targeting of all three homeologs like this example
Dsubgenome will act like a homozygote but will be biallic due to two different cuts at different times

82
Q

What are you looking for in a screen T1 generation and segregation?

A

Resulting T0 plant - heterozygous mutation and hemizygous transgene
Select for T1 segregant - Homozygous mutation and no transgene
Diploid plant occurs in a 3:1 ratio in wheat 1:64 chance for homozygous edit

83
Q

What are examples of class 1 CRISPR system and the bacteria its from?

A

I-C - Bacillus halodurans
I-U - Geobacter sulfurreducens
I-E - Escherichia coli
III-B Pyrococcus furiosus

84
Q

What are examples of class 2 CRISPR system and the bacteria its from?

A

V-U3 - Bacillus thuringiensis
VI-B1 - Prevotella buccae
II-A - Streptococcus thermophilus

85
Q

What is an overview of CBE and ABE?

A

CBE: Cytidine base editors makes C to T (or G to A) changes
ABE: Adenine base editors makes A to G (T/C) changes
Cas9n protein (no DSB)
PAM needs to be very close, PAM motif on different side

86
Q

What is prime editing?

A

The PE system retains CRISPR’s targeting specificity but carries with it, additional cargo in the form of an edit-containing RNA template as a contiguous extension of the guide RNA (known as the pegRNA)
And uses reverse transcriptase to create new sequence
Only 30bp can be edited but 2 prime editors at once result in 60bp sequences

3s2: Plant biotechnology (9 decks)

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