EB20

Question 1

How many deaths per year from Malaria

Answer 1

100s of thousands
mostly infants
mostly in Africa

Question 2

what are current interventions for malaria and how effective are they
- whats the most optimistic scenario

Answer 2

nets spraying, drugs, saved millions but not enough to eliminate, resistance may lead to reversal and cost more than funding available.
most optimistic scenario (cost up to 9 billion a year) still leaves malaria in 62 countries by 2030

Question 3

what are current innovations for malaria treatment

Answer 3

diagnostics, drugs, (one dose cures), vaccines, (75% efficiency allowing 80% coverage, implied 60% transmission reduction) and insecticides to maintain current levels in face of resistance will not be enough for eradication

Question 4

what is the biological background of malaria

Answer 4

1. Largely in rural africa
2. 4 spp. of plasmodium causing: falciparum, vivax, malariae adn ovale.
3. most transmission in Africa by A. gambiae/colluzii, arabiensis and funestus.
4. species can also transmit O’nyong’nyong virus and filiariasis.
5. vast maj of mosquitos dont transmit disease, only females bite and transmit

Question 5

what could be two uses of gene drive in malaria

Answer 5

population suppression: reduce mosquito no.

population replacement: change mosquito so can no longer transmit

Question 6

How are trasngenic mosqutios made

Answer 6

1. plasmid and helper RNA micro injected into freshly laid eggs with a fine needle.
2. many eggs die but some survive and incorperate DNA into their genome,
3. injected adults are crossed to WT, larvae screen for RFP to identify transgenic

Question 7

What is population suppression: Sex ratio bias of mosqutios theory

Answer 7

1. based on naturally occuring driving Y chromosome in Aedes. - visible in crossing experiments
   —–when male is making sperm at meiosis breaks down X
2. gradually convert population so it is predominantly male: dont bite so dont transmit.
3. experiments make 95% males with normal fertility.
   only functional sperm will carry y chromosome = males progeny.

Question 8

Describe the method of Y chromosome drive in Anopheles (IC research)

Answer 8

1. enzyme made to cut X specific sequence (.e.g ribosomal repeat found only on X, in 100’s of copies).
2. HEG coding Ppol enzyme inserted into Y chromsoome region - with control region flanking enzyme (beta 2 tubulin promoter), which is only expressed at meiosis so gene only expressed then.
3. enzyme labelled wth GFP.

Question 9

what did the trials of the IC anopheles Y chromosome drive show
how was it fixed

Answer 9

1. first attempt didnt work as instead of making fertile males with a male sex bias, made sterile males.
2. Enzyme made in testes was incorperated intt sperm and deliverred to zygote which cut up X chromsoome in zygote.
   - fixed by reducing the half life of the enzyme so wouldnt be active in zygote.

Question 10

Describe gene KO by homing

Answer 10

• can be used to gradually target gene in mosquito
  1. cell heterozygous for homing endonuclease gene makes an enzyme that cuts recognition site for HEG in chromosome, uses homologous chromsoome as template for repair and gene gets copied across, so heterozygote goes to homozygote.
  2. recognition site is only found in chromosome not containing the gene, the gene is in the middle of its own recognition site.
  3. nuclease gene can be spread through the population by same method as HEG and recognition site is a fertility gene.
  4. HEG will cause population wide KO of target gene.

Question 11

where are HEGs found

Answer 11

found anturally in fungi, algae, protists and viruses but not animals.

Question 12

describe the 3 basic steps in homing KO of fertility genes

Answer 12

1. HEG males released and transmit HEG to most progeny
2. mating of carriers prooducing homozygotres.
3. homozygous male is fertile and continues to transmit HEG whereas homozygous female is sterile.

Question 13

first demonstration of homing in animals

Answer 13

1. taking homing endonuclease gene and recognition site from yeast.
2. strong deviation from 50:50 inheritances, spread through a small cage population.
3. high CRISPR based homing rates at female fertility genes. 94% across all genes.

Question 14

What three endonucleases have been trialed to target mosquito genes

Answer 14

1. TALENs
2. HEG
3. CRISPR/Cas

Question 15

describe TALEN endonuclease

Answer 15

• Fok1 cleavage domain can be linked to site specific recognition domain in TALENs: easy to engineer, binding/cleavage sites dont overlap, repetative structure.

Question 16

describe HEG endonuclease

Answer 16

highly specific, found in primitive organiss, can be re-engineered to target DNA. efficient and highly specifci naturally select to home binding cleavage sites overlap, takes time to re-engineer.

Question 17

describe CRISPR/Cas endonuclease

Answer 17

can be guided to its target site by a short guide RNA

easy to make enzyme that can recognise and cut a sequence of DNA> binding cleavage sites overlap.

Question 18

what is the time course to elimination predicted

Answer 18

Expected 3-4 years after release

Question 19

Describe the TARGET MALARIA technology

Answer 19

• product is a bucket of male mosquitos carrying a gene coding for a DNA nuclease
• genes will spread through the target population over time and suppress/modify populations to reduce vectorial capacity and malaria transmission.
• reducing burden of disease and facilitate other elimination programmes

Question 20

What are the intended key features of TARGET MALARIA technology

Answer 20

1. reduce transmission.
2. widely applicable: urban/rural, high/low disease, indoor/outdoor biting, irresepctive of available health system, stability of government, location of communities (access).
3. area wide and regional control, long lasting compatible with and helpful to other interventions; no cross resistance.
4. safe and inexpensive.

Question 21

What is a biological precedent for mosquito gene

Answer 21

control of Cassava mealybug.

1. Cassava is a stable crop to SA grown widely in Africa
2. bug came over from SA and caused devastation.
3. Parasitoid wasp that only attacks mealy bug released.
4. Large redution in bug population in short time.
5. overall benefit cost ratio was good.
6. self spreading nature as in gene drive, biological control

Question 22

Why may mosquito gene drive not work

Answer 22

males wont compete with WT males

• replenish lab stocks with natural varaition to keep it close to whats our there. = soluble problem.
• genetic construct itself is unlikley to cause problems as it is only switched on in testes.

Question 23

potential harms if gene drive does work

Answer 23

1. possible extinction of mosquito species
2. disruption of ecosystem
3. empty niche filled by other vectors for disease
4. plasmodoum evolves to other vectors
5. mutations in nuclease gene after release.
6. Resistance to mutation

Question 24

How do mosquitos mate in nature

Answer 24

males form swarms and and females fly into them, choose a mate, then fall out of swam. males are only alive for 4-5 nights.

Question 25

what is the harm in possible exctinction of mosquitos

Answer 25

moral | likely a population will be kept in the lab so wont ever be fully extinct, captive breeding programme.

Question 26

how could mosquito gene drive disrupt ecosystem

Answer 26

Pollinator: sweep net to see whether they have pollen, care mostly if its a specialist pollinator. Prey: examine gut content or feaces of suspected predators, take samples and do DNA barcoding of region of mtDDNA and amplify by PCR. - see what fraction diet is due to mosquito species. ** can test manipulatively impose suppression and see what happens. ** nobody has currently reported ecological consequenes

Question 27

implications of empty niche being filled by something else

Answer 27

* malaria is prevelent in Africa can be related to efficiency vectors: several years ago A. arabiensis accdientally intro to SA. - more efficient than local mosquito leading to outbreak. * potentialy african vectors better as evolved with humans longer. * can therefore nassume that if african vectors eliminated SA vectors would not fill their niche as not as good. eliminating the worst, means nothing worse can follow.

Question 28

what is the implication of plasmodium evolving to other species/organisms

Answer 28

lab selection experiment seems unlikely people are bittne all the time by culex and aedes so any mutant that can transmit plasmodium would give plasmodium a selective advantage, but has not occured in nature.

Question 29

what are the implications for mutations in nuclease gene after release

Answer 29

``` function KO is not a concern other sorts of mutation: mutate insert into region dont want, e.g. insertion in immune system could make them better vectors. ```

Question 30

what are the implications for resistance to gene drive mutation

Answer 30

1. mutations in target site no longer recognised by endonucklease 2. for resistance: target essential sequence so cant change and still maintain function 3. could have multiple guide RNAs targetting three different places 4. could get selection for things not recognised by the enzymehttps://www.brainscape.com