Genetic Manipulation Technology 5&6 Flashcards
dodName 3 methods of genome
editing
Zinc finger nucleases
Talens
Cas9/CRISPR
What do these genome editing methods actually do
They find ways to direct a DNA cleaving enzyme to specific sites in any genome where you want to make mutations
What are zinc-finger nucleases
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
What does FOK1 do
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
What is non-homologus end joining (NHEJ)
Repairs by DNA enzymes will usually cause errors - causing point mutations - 70-80% of time (insertions or deletions)
Why are ZFN good
Work on any animals
Work in vitro and vivo, with good specificity
What are TALENS
Transcription Activator-Like Effector Nucleases
How do TALENs work
Work similar to ZFNs
comprise of nonspecific FOK1 nuclease domain and customizable DNA binding domain on both sides to cut double stranded DNA
What do TALENs look like
33-35bp repeats - easily recognisable
How do TALENs cause mutations
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
What is Cas9
Double-stranded endonuclease - cuts DNA on both strands with one cas9
Where does Cas9 come from
Bacterial acquired immunity against virises
What is CRISPR
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.
What does Cas-mediated mutagenesis stand for
CRISPR associated mutagenesis
What are the two parts of Cas-mediated mutagenesis and their roles
CRISPR-RNA
Transactivation CRISPR-RNA
These RANA’s guide Cas9 to the specific site to cut gene and mutation is caused through repair
Where can Cas-mediated mutagenesis only occur
Needs to be adjacent to a protospacer adjacent motif (2-5bp that are NGG)
How does cas-mediated mutagenesis/CRISPR work
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
what DNA can CRISPR cut out?
and what is the base pair for Cas9?
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’
How is CAS9 made artificially to work in genes
A section of DNA is added to cells which include the CAS9 gene and crRNA and tracrRNA
What is the difference between CRISPR/CAS in the wild and in the lab
Wild - two separate sections (crRNA and tracrRNA)
Lab - crRNA and tracrRNA fused
Why is CAS9/CRISPR the most popular
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
What is one disadvantage of the CAS9/CRISPR system
Mutations induced are uncontrolled and variable
What is homology directered repair (in CRISPR)
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
How are animals cloned
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
How is the clone oocyte activated
By placing oocytes in strontium chloride, this is because it causes calcium waves which activate growth.
Electrical stimulation can do the same
Why is cloning of interest
Endangered species
Pets
Clone people
Study biology (ageing etc)
Therapeutic cloning (spare organs)
Why is cloning useful for animals
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
What are the issues of clone animals
Unhealthy - large offspring, die early, premature ageing, liver failure, weak immune system, respiratory issues
What causes the issues of cloned animals
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
How can cloning be useful to human medicine
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.
Give some technical challenges of stem cell technology
Clinical grade culture facility
Prevent spontaneous differentiation (start of fetus)
To overcome recipients immune response
To produced cell types that are needed only
How did Takahashi and Yamanaka make pluripotent stem cells from mice embryos and human fibroblasts
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
How did Takahashi and Yamanaka confirm these cells were pluripotent
Teratoma formation under mice skin
Embryoid bodies in tissue culture
Blastocyst implantation forms if implanted into mice uterus
Define iPS
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.
How does iPS work
Unknown for sure but Oct4, sox2 and Nanog is needed to maintain pluripotency
What did Kim et al. find regarding epigenetic memory in induced pluripotent stem cells
Easier to change them back to OG cell suggesting some memory stays
What did Feng et al find regarding iPS cells efficacy in comparison s to make embryonic stem cells
iPS cells were less likely to form haemangioblasts, more apoptosis and severely limited growth (1000 times less) than embryonic stem cells.
What are the ethical considerations regarding genetic modification in humans
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
Why is iPS a worry for causing cancer
Okita et al. - 2007
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.
What did Tateishi et al. do in 2008 regarding iPS and diabetes
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.
