Genetic Manipulation Technology 5&6

Question 1

dodName 3 methods of genome
editing

Answer 1

Zinc finger nucleases
Talens
Cas9/CRISPR

Question 2

What do these genome editing methods actually do

Answer 2

They find ways to direct a DNA cleaving enzyme to specific sites in any genome where you want to make mutations

Question 3

What are zinc-finger nucleases

Answer 3

Protein motif that binds to 3’ base end - every zinc finger has a codon that is specific to each 3 base pairs - a chain of base pairs will bind to specific sequences and transcribed.

-> Zn-finger transcription factor binds specific consensus DNA sequences around genes and activates transcription. Different Zn-finger transcription factors with different DNA binding specificities control the regulation of different

Question 4

What does FOK1 do

Answer 4

A single-stranded DNA nuclease - FOK1 cuts the DNA strand and the ZFN cuts the other side (done on both strands) –> creates a double-stranded DNA break

Question 5

What is non-homologus end joining (NHEJ)

Answer 5

Repairs by DNA enzymes will usually cause errors - causing point mutations - 70-80% of time (insertions or deletions)

Question 6

Why are ZFN good

Answer 6

Work on any animals
Work in vitro and vivo, with good specificity

Question 7

What are TALENS

Answer 7

Transcription Activator-Like Effector Nucleases

Question 8

How do TALENs work

Answer 8

Work similar to ZFNs
comprise of nonspecific FOK1 nuclease domain and customizable DNA binding domain on both sides to cut double stranded DNA

Question 9

What do TALENs look like

Answer 9

33-35bp repeats - easily recognisable

Question 10

How do TALENs cause mutations

Answer 10

Non-homologous end joining

step by step:
1. 2 talens TALENs binding specific sequence of target gene to create double strand break
2. 2 hypervariable amino acids determine which base each TALE repeat binds to
3. A TALE binding its target DNA sequence

Question 11

What is Cas9

Answer 11

Double-stranded endonuclease - cuts DNA on both strands with one cas9

Question 12

Where does Cas9 come from

Answer 12

Bacterial acquired immunity against virises

Question 13

What is CRISPR

Answer 13

Clustered regularly interspaced short palindromic repeats - essentially means equally spaced repeats of DNA bases with coding DNA in between related to pathogens. Used in bacteria to identify bacteriophages/viruses in the past. When this coded DNA is identified from the pathogen it is matched up with Cas9 –> cleaved and removed to allow cas-mediated mutagenesis to occur thus killing the pathogen.

Question 14

What does Cas-mediated mutagenesis stand for

Answer 14

CRISPR associated mutagenesis

Question 15

What are the two parts of Cas-mediated mutagenesis and their roles

Answer 15

CRISPR-RNA
Transactivation CRISPR-RNA

These RANA’s guide Cas9 to the specific site to cut gene and mutation is caused through repair

Question 16

Where can Cas-mediated mutagenesis only occur

Answer 16

Needs to be adjacent to a protospacer adjacent motif (2-5bp that are NGG)

Question 17

How does cas-mediated mutagenesis/CRISPR work

Answer 17

1,The first CRISPR crRNA is complementary to part of the target gene, upstream of the PAM.
2.Go into DNA duplex and binds by base-pairing
3. The transactivating CRISPR tracrRNA () has a region that is complementary to the rest of the crRNA
4.It binds by base-pairing
5. A Cas9/crRNA/tracrRNA complex binds the target sequence
6.Cas9 will make a double-stranded break in the target DNA, a variable number of bases from the PAM. Break will be ragged, with single strand overhangs

BUT….
If you introduce Cas9 and crRNA and tracrRNAs into eukaryotic cells, it will work to cleave the targeted DNA
But rather than killing the cell, DNA repair enzymes will try to repair the damage
They will screw it up, creating a mutation

crRNA - forms a DNA duplex by binding to the coded section of its 5 end to the matching section of the DNA - the DNA will bind to the RNA rather than its own strand.
The other half of the crRNA is NOT complementary to the DNA strand after the PAM sequence, but the tracrRNA IS complementary to the part of the crRNA which isn’t bound to DNA

This triggers CAS9 to come over and make a double break at the crRNA. The DNA repair enzymes come in to fix it however they don’t know what is missing - makes mistakes/mutations e.g. non-homologous end joining

Question 18

what DNA can CRISPR cut out?

and what is the base pair for Cas9?

Answer 18

Cas/CRISPR system can cut any piece of genomic DNA that is adjacent to a ‘PAM’ (Protospacer adjacent motif)
These are 2-5 bp sequences that differ between bacterial species

Base pair for for Cas9 is ‘NGG’

Question 19

How is CAS9 made artificially to work in genes

Answer 19

A section of DNA is added to cells which include the CAS9 gene and crRNA and tracrRNA

Question 20

What is the difference between CRISPR/CAS in the wild and in the lab

Answer 20

Wild - two separate sections (crRNA and tracrRNA)

Lab - crRNA and tracrRNA fused

Question 21

Why is CAS9/CRISPR the most popular

Answer 21

Only have to make DNA not proteins
Also affects both alleles not just one
Can do more than one gene at a time
Can inject into zygotes - so a lot quicker than homologous recombination from about 9 months to about 4 weeks

Question 22

What is one disadvantage of the CAS9/CRISPR system

Answer 22

Mutations induced are uncontrolled and variable

Question 23

What is homology directered repair (in CRISPR)

Answer 23

Tells the DNA repair enzymes what sequence they have to put in to stop random mutations - can be a whole strand or a single oligonucleotide
Can also add loxP sites for gene knock out

Homology-Directed repair can also be used with TALENs and zinc-finger nucleases

Question 24

How are animals cloned

Answer 24

Through somatic cell nuclear transfer :

Take a cell from a donor animal –> take the nucleus out of recipient unfertilised oocyte –> put the nucleus of the donor somatic cell into the enucleate oocyte (direct injection/electrofusion) –> activate the oocyte to start development (is reprogrammed by cytoplasm of host to become totipotent again) –> transfer it the the uterus of pseudopregnant female –> completes development as normal in utero

Question 25

How is the clone oocyte activated

Answer 25

By placing oocytes in strontium chloride, this is because it causes calcium waves which activate growth.
Electrical stimulation can do the same

Question 26

Why is cloning of interest

Answer 26

Endangered species
Pets
Clone people
Study biology (ageing etc)
Therapeutic cloning (spare organs)

Question 27

Why is cloning useful for animals

Answer 27

Mice are genetically identical but farm animals are not therefore this allows a method to make transgenic farm animals - allowing studies to be done on them, similar to mice

Question 28

What are the issues of clone animals

Answer 28

Unhealthy - large offspring, die early, premature ageing, liver failure, weak immune system, respiratory issues

Question 29

What causes the issues of cloned animals

Answer 29

Failed programming from the differentiated cell from the original animal
Accumulation of damaged macromolecules - because they’re derived from an old cell
Shortened telomeres - DNA eventually unravels
Retention of environmental mutations from donors cell so increased mutational load from donor

Question 30

How can cloning be useful to human medicine

Answer 30

Take a donor cell and inject into an embryo and let it grow to a blastocyst and take out the blastocyst stem cells to make heart precursors, pulmonary cells etc…

This helps to avoid the issues spoken about earlier in the cloning of animals where the cloned animals are unhealthy and die young. This is because it closely simulates what happens to a fetus in vivo.

Question 31

Give some technical challenges of stem cell technology

Answer 31

Clinical grade culture facility
Prevent spontaneous differentiation (start of fetus)
To overcome recipients immune response
To produced cell types that are needed only

Question 32

How did Takahashi and Yamanaka make pluripotent stem cells from mice embryos and human fibroblasts

Answer 32

Infected mice with retroviruses expressing the 24 genes needed for embryonic stem cell development
Eventually, they found that 4 of these genes can cause pluripotency without the other 20

Question 33

How did Takahashi and Yamanaka confirm these cells were pluripotent

Answer 33

Teratoma formation under mice skin
Embryoid bodies in tissue culture
Blastocyst implantation forms if implanted into mice uterus

Question 34

Define iPS

Answer 34

Induced pluripotent stem (iPS) cells, are a type of pluripotent stem cell-derived from adult somatic cells that have been genetically reprogrammed to an embryonic stem (ES) cell-like state through the forced expression of genes and factors important for maintaining the defining properties of ES cells.

Question 35

How does iPS work

Answer 35

Unknown for sure but Oct4, sox2 and Nanog is needed to maintain pluripotency

Question 36

What did Kim et al. find regarding epigenetic memory in induced pluripotent stem cells

Answer 36

Easier to change them back to OG cell suggesting some memory stays

Question 37

What did Feng et al find regarding iPS cells efficacy in comparison s to make embryonic stem cells

Answer 37

iPS cells were less likely to form haemangioblasts, more apoptosis and severely limited growth (1000 times less) than embryonic stem cells.

Question 38

What are the ethical considerations regarding genetic modification in humans

Answer 38

Not allowed except the exception of mitochondrial replacement
Better targeted gene therapy for muscular dystrophy and sickle cell anaemia
Slippery slope to eugenics
Gene therapies that aren’t inherited are more accepted - used for SCID

Question 39

Why is iPS a worry for causing cancer

Okita et al. - 2007

Answer 39

c-Myc over-expression may cause tumor development after transplantation of iPS derived cells. the chimeras and progeny derived from iPS cells frequently showed tumor formation. They found that the retroviral expression of c-Myc was reactivated in these tumors.

Question 40

What did Tateishi et al. do in 2008 regarding iPS and diabetes

Answer 40

demonstrated that insulin-producing islet-like clusters (ILCs) can be generated from the human iPS cells under feeder-free conditions. The iPS cell derived ILCs not only contain C-peptide positive and glucagon-positive cells but also release C-peptide upon glucose stimulation.