GM plants and Gene Editing

Question 1

What is the definition of GMO?

Answer 1

An organism in which the genetic material has been altered in way that does not occur naturally by mating and/or natural recombination

Question 2

What are the most popular GM crops?

Answer 2

Canola/oilseed rape
Cotton
Maize/corn
Soybean

Question 3

Which gm crops are authorised in the EU and what are they used for?

Answer 3

Soya bean (pest resistant/herbicide tolerant/modified fatty acid profile)- Food and food additives

Maize (pest resistant/herbicide tolerant/drought tolerance)- Food and food additives

Oilseed rape (herbicide tolerant)- Food and food additives

Cotton (pest resistant/herbicide tolerant)- Food and Food additives

Question 4

Why are GM food labels important?

Answer 4

Food labels are essential for consumers to make a choice
A specific GM label is required if more than 0.9% of a food or ingredient is derived from a GM source

Question 5

What is a Agrobacterium tumefaciens infection?

Answer 5

When the microbe infects a plant and inserts its DNA, this forces the plant to produce hormones that multiply the cells and allows the pathogen to feed. Agrobacterium tumefaciens live inside the galls

Question 6

What is Agrobacterium tumefaciens?

Answer 6

A pathogen that genetically modifies plants

Question 7

How is Agrobacterium tumefaciens used in the lab?

Answer 7

The plasmid of the agrobacterium tumefaciens to transfer the DNA of choice into a plant cell

Question 8

What are the common traits of GM plants?

Answer 8

Herbicide resistance
Insect resistance
Nutritional traits

Question 9

How popular is Herbicide resistance as a GM trait?

Answer 9

Widespread use of glyphosate herbicides on crops such as soybeans, cotton, and tobacco since the late 1980’s
About 80% of the U.S market in farm crops is now in plants that tolerate glyphosate
Herbicide resistance is the most widely planted transgenic crop trait

Question 10

What is Glyphosate?

Answer 10

It is a herbicide that inhibits EPSPS.
EPSPS is an enzyme that is important in aromatic amino acid production (phenylalanine, tryptophan and tyrosine). A bacterial EPSPS is naturally resistant to glyphosate.

Question 11

What is the process of getting a Glyphosate tolerant plant?

Answer 11

1. Clone the resistant EPSPS from bacteria and insert it into a vector (plasmid DNA circle)
2. Transfer the vector into the plant. Use of chloroplast targeted peptide for EPSPS
3. With the bacterial form of EPSPS, you can then spray with glyphosate
4. The EPSPS form naturally found in plants in inhibited, and only the plants with the bacterial form can survive the glyphosate application
5. Plants not modified with the vector containing the bacterial EPSPS (i.e weeds) die

Question 12

What is Bacillus thuringiensis (Bt)?

Answer 12

It is a soil bacterium commonly used as a biological pesticide
Bt toxin is specific to receptors in insect gut wall

Question 13

How does Bt work?

Answer 13

• Bt gene is transferred from the bacillus into corn
• European corn borer feeds on the corn plant and ingests the protein encoded by the Bt gene
• The Bt protein penetrates and collapses the cells lining the gut and the insect dies

Question 14

What is the mode of action of Bt?

Answer 14

The Bt toxin binds to specific receptors called cadherins on the brush border of the gut cells. Toxin binding to cadherin proteins results in formation of toxin oligomers on regions of the cell membrane
Accumulation of toxin oligomers results in toxin insertion in the membrane, forming a pore and ultimately insect death.

Question 15

How are Bt proteins shaped?

Answer 15

Bt proteins are shaped like crystals, so they are commonly called ‘crystalline toxins’ or ‘cry toxins’
Once digested the protein is activated and then binds to specific receptors in insect guts. Once bound, the Cry toxins pierce holes in the insects gut, ultimately causing the contents to leak and the insect to starve.

Question 16

How do Cry toxins pass through humans with no effect?

Answer 16

Humans do not have the same receptors or gut conditions as insects

Question 17

What is an example of a nutritional trait in GM plants?

Answer 17

Golden rice
Genes which encode enzymes to make beta-carotene (precursor to vitamin A) were introduced to rice from daffodil and bacteria

Question 18

What is another example of a nutritional trait in GM plants?

Answer 18

Purple tomatoes
The Anthocyanin pathway is controlled by several genes encoding enzymes for each step
Therefore, difficult to genetically engineer as there are many steps and enzymes

Question 19

How are transcription factors used to control gene expression?

Answer 19

We can use expression of transcription factors to drive downstream gene expression, in this case genes which encode enzymes in the phenylpropanoid pathway for anthocyanin

Question 20

What is an example of transcription factors being used for gene expression?

Answer 20

The greatest enrichment in anthocyanins has been engineered in tomato by combining expression of the R2R3MYB protein (Rosea 1) and bHLH (Delila) from snap dragon.
These are both transcription factors

Question 21

What is Cisgenesis?

Answer 21

Cisgenesis is genetic modification to transfer beneficial alleles (gene version) from crossable species into a recipient plant
The donor genes transferred by cisgenesis are the same as those used in traditional breeding.
Genes are only transferred between closely related organisms

Question 22

What is a Cisgenic example?

Answer 22

Downy mildew resistance genes from wild grapevine (M. rotundifolia) into commercial cultivars of Vitis vinifera

Question 23

What is genome editing?

Answer 23

Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or molecular scissors

Question 24

What are the two category regulations implemented by the EU?

Answer 24

Category 1 plants exempt from GMO legislation. These could occur naturally or via conventional breeding. As long as no endogenous gene is disrupted and the inserted DNA already exists in the breeders gene pool.
Any other insertions should not be longer than 20 nucleotides.

Category 2- plants that fall outside this scope would be known as category 2. Remain under current GMO legislation

Question 25

What does Genome editing rely on?

Answer 25

DNA repair

Question 26

What are meganucleases?

Answer 26

Recognize large targets and encoded by mobile genetic elements Large recognition site dsDNA sequences of 12 to 40 base pairs

Question 27

What are ZFNs?

Answer 27

Zinc-finger nucleases (ZFNs) are generated by fusing a zinc finger DNA-binding domain to Fok1 from Flavobacterium okeanokoites, an endonuclease They can target specific DNA sequence

Question 28

What do ZFNs do?

Answer 28

Artificial restriction enzymes. Engineered zinc-finger (ZF) DNA binding domains fused to a nonspecific nuclease domain, so called ZF nucleases (ZFNs) Customised zinc-finger nucleases (ZFNs) can create a DNA double strand break (DSB) at preselected sites in the genome.

Question 29

What are TALENs?

Answer 29

DNA binding proteins discovered in Xanthomonas bacteria Transcription activator like (TAL) effector proteins from Xanthomonas spp. fused to Fok 1 from Flavobacterium okeanokoites, an endonuclease

Question 30

How do TALEN proteins work?

Answer 30

Transcription activator like (TAL) effector proteins are produced by bacteria in the genus Xanthomonas Natural TAL bind to specific sequences of host DNA and have an effector protein Specifically modified TAL DNA-binding protein domains can be linked to a custom effector domain (e.g., a nuclease, or a transcription activator or repressor) to create a chimeric protein capable of precisely targeted DNA manipulation

Question 31

What are the Xanthomonas spp.?

Answer 31

Xanothomonas spp. are plant pathogens which uses TAL effectors to manipulate gene expression in the host plant cell

Question 32

What is CRISPR/Cas?

Answer 32

Can be used for mammalian cell genome editing CRISPR-Cas can be used as molecular scissors to cut at specific locations in the genome. Subsequent repair of the cut by the cells endogenous repair mechanism can introduce changes at the specific cut site.

Question 33

How is Crispr-Cas used in bacteria?

Answer 33

Many bacteria protect themselves from invading viruses by using adaptive immune system called CRISPR-Cas Spacers are transcribed into short RNA guides that match regions of the viral genome CRISPR-cas systems can recognize genetic sequences specific to that of viral invaders and cut them

Question 34

What is a key component of the crispr-cas gene editing system and what is it made up of?

Answer 34

The gRNA is made up of two parts: 1. crispr RNA (crRNA) a 17-20 nucleotide sequence complementary to the target DNA 2. tracr RNA, which serves as a binding scaffold for the Cas nuclease

Question 35

What is an example of a gene edited Crispr plant?

Answer 35

Genome-edited mushroom have small deletions in a specific gene but contain no foreign DNA integration in its genome

Question 36

How is Crispr-Cas used to create gene edited mushrooms?

Answer 36

Crispr-cas9 technology to create small deletions in a specific gene encoding a polyphenol oxidase (PPO) enzyme polyphenol oxidase which causes browning to occur A bacterial plasmid construct to deliver the guide RNA and Cas9 enzyme into mushroom cells to achieve the necessary deletions

Question 37

What is Gene Therapy and Gene editing?

Answer 37

Gene therapy- The therapeutic delivery of genetic material to a person with the intention of compensating for a genes absence or dysfunction Gene editing- the permanent addition, removal, or correction of genetic material within the genome

Question 38

Can gene editing be a type of gene therapy ?

Answer 38

Yes if they both target the cause of disease, such as a variant or mutation in a gene, by using genetic material to treat or prevent disease

Question 39

What are used to construct the capsule of Cas9 mRNA and sgRNA?

Answer 39

lipids

Question 40

What is PLK1?

Answer 40

Polo-like kinase 1 plays an important role in the initiation, maintenance and completion of mitosis