W5L2 Gene driver

Question 1

What is a gene drive

Answer 1

• An inheritance that does not act in a Mendelian way
• A bias inheritance
  -can lead to skewing of phenotype or genotype down the germ line

Question 2

Natural gene drive in mice

Answer 2

-A t-haplotype is a natural occurring gene (13-55%)
-up to 95% is transmitted in male
-but does not fix, probably due to fitness cost
- We can tweek it with CRISPR/cas9 trans gene to targets female fertility gene can cause population crash
-200k crash in 28 years

Question 3

CRISPR / Cas9 gene-editing technology

Answer 3

Small guide RNA molecules take the Cas9 endonuclease enzyme to specific nucleotide sequences in the genome to cut a section of DNA
- dSDNA is then repairs either by non-homologous end joining or homologous repair (from a homologous template or sister chromatin or with transgene)

Question 4

what if you repair the cut DNA with genes encoding the machinery that can cut

Answer 4

-we can spread it down a germ line. Man-made selfish elements
-able to make a mutagenic chain reaction, creating a homozygous in one generation

Question 5

‘Homing Endonucleases’ for Gene drives in natural organism

Answer 5

• Recognize a sequence and insert into it to a particular area.
• Occur naturally in fungi, plants,bacteria, bacteriophage
• Often associated with self-splicing intron

Question 6

Application for gene drive

Answer 6

A commonly used example is driving malaria or dengue resistance genes through Mosquito populations
-affect the fertility of invasive species
-knock out the toxin producing gene of cane toad

Question 7

type of gene drive by objective

Answer 7

• modification drive (anti-malaria resistance in mosquitoes)
  suppression drive: crashing wild pop

Question 8

Gene drive by mechanism

Answer 8

-homing drive
-toxin-antidote drive
-shredder drive
-manipulation using an endosymbiont

Question 9

Homing drive

Answer 9

-after an insertion of transgene/ gene drive cassette (sg RNA, Cas9 and Cargo), it will be translated
-sg RNA guide the Cas9 to cleave target DNA
-cleave repair using the trans gene as template

Question 10

Toxin antidote drive background information

Answer 10

Naturally occurring Medea (Maternal-effect dominant embryonic arrest) elements of Tribolium castaneum exhibit segregation distortion
- in a M/m mother, m/m would fail to develop
-try to transfer it to Drosophila/mosquitos

Question 11

Toxin/antidote indept

Answer 11

-media construct have a toxin and an antidote. Having both is fine, toxin only is bad
- toxin create miRNA which target mRNA
- in fly, also pass toxin mRNA, kill wild type
-zygotes antidote can prevent maternal mRNA

Question 12

Driving Y’s (X-shredder)

Answer 12

• CRISPR/cas9 cut the X chromosome. Shredded X are inviable
• Only Y-bearing sperm produce viable offspring
• So fathers only have sons
• Sex ratio bias causes population crash

Question 13

Prerequisite of X-shredder

Answer 13

• Construct on the Y carries X-shredder sgRNA
• Spermatogenesis promoter

Question 14

Haplolethal loci on the X Chromosome

Answer 14

• Over >10,000 genes in the Drosophila have lethal alleles
• > 750 have been located on the X
• The vast majority of them are recessive
• If a fly needs two functional copies to survive are called ‘haplolethal’ loci.
• There are 11 haplo-lethals on the X chromosome

Question 15

Sex skewing by targeting Haplolethal loci on the X

Answer 15

-premeiotic germ line Cas9 activity expressed from autosome
-generation of null allele at haplolethal X
-meiosis and sperm generation, gene drive is inherited
-fertilization of wildtype egg lead to post-zygostic elimination of female offspring

Question 16

reverse sweep

Answer 16

-by adding a transgenic w/ insecticide susceptibility into a population that have resistance, the future generation would now loss their resistance
-but it’s unlikely to be fixed
-can lead to lower insecticide
-can try to have a transgene susceptible to a normally non harmful substance

Question 17

What is screw worm fly

Answer 17

• Screw worm fly, which kills cattle, eliminated from USA

Question 18

Genetic control of Screw worm with sterile male release

Answer 18

• 1960’s technology
• 500 million screw worms per week on artificial diets
• Males irradiated ->chromosomal damage and sterility
• Dropped from aeroplanes
• Sterile males far-outnumbered wild males
• Mated with females and embryo’s died during development

Question 19

Problem with gene drive

Answer 19

• Not effective because new mutations or standing variation is resistant to the engineered trait or to the drive itself
• Spread to non-target populations or organisms via Lateral
  Gene Transfer or Hybridization or cross species interbreeding
• Side effects on targeted organism?
• Will it reduce genetic diversity?
• Extends beyond intended range
• Ecological risks!!!
• Ethical?