Week 9: Playing with the genome Flashcards
Definition of a genetically modified organism (GMO)
Modifying an organism by adding DNA sequences or genes to a particular organism that are not normally found within that organism (eg. tomatoes, bananas and strawberries)
Ethical concerns regarding GMOs
- Unsure of long term impacts
- Misuse (to control a population)
- Agriculture - farmers may get locked into using these technologies by the companies that produce the technology
Transgenic organism
- An organism containing foreign DNA
Artificial selection of carrots
- Root size (more to eat)
- Colour (to distinguish from wild carrot)
What is recombinant DNA technology?
- Recombinant DNA technology involves using enzymes and various laboratory techniques used to manipulate and isolate DNA segments of interest. This method can be used to combine or splice DNA from different species or to create genes with new functions. The resulting copies are often referred to as recombinant DNA.
Molecular cloning
- Need a way to copy your sequence of interest
- ‘Clone it’ into a bacterial chromosome
- Transform bacteria to express your cloned sequence
Molecular cloning in the production of insulin
- Plasmids
- Restriction enzymes - to guide the cut
- DNA Ligase - stick DNA back together
- PCR
Steps of molecular cloning
You have the gene of interest which is insulin, use PCR to identify the sequence of interest, use the restriction enzyme to cut the sequence so that it has the correct overhangs at the end. Can stick the insulin gene back into the plasmid to make recombinant DNA
Modern tools that theoretically make molecular cloning much easier
- UCSC genome browser / NCBI sequence data / Ensembl
- PCR techniques
- Engineered plasmid vectors
- Modified restriction enzymes for cutting specific DNA sequences
- Compliment bacterial stains
Why make a gene knockout (KO) mouse?
- To mimic what happens in a human, for example the function of a particular gene in vivo
- To study gene function
- What happens to the phenotype of the mouse if the gene is deleted
Definitions of the word ‘phenotype’
The observable characteristics of an individual resulting from the interaction of their genotype with the environment
Definition of ‘stem cell’
Undifferentiated cell that has the ability to specialise into any cell type
Homologous recombination
Exchange of genetic code between identical/similar (homologous sequences)
* Natural repair mechanism
* Can be used to insert desired DNA sequences
General strategy for gene targeting in mice
- Embryonic stem (ES) cell culture
* ES cells are cultivated from mouse pre-implantation embryos (blastocysts) - Construction of targeting vector
* The vector contains pieces of DNA that are homologous to the target gene, as well as inserted DNA which changes the target gene and allows for positive-negative selection - ES cell transfection
* The cellular machinery for homologous recombination allows the targeting vector to find and recombine with the target gene. - Proliferation of targeted ES cell
* Selection of presence of neo+ and absence of HSV enriches targeted ES cells. - Infection of ES cells into blastocysts
* The targeted ES cells are infected into blastocysts where they mix and form a mosaic with the cells of the inner cell mass from which the embryo develops. The infected blastocysts are implanted into a surrogate mother where they develop into embryos. - Birth and breeding of mosaic mice
* The mosaic mice mate with normal mice to produce both gene targeted and normal offspring
Summary
* Design a target vector
* Homologous recombination in ES cells
* Screen ES cells
* Infect cells into blastocysts
* Implant into female mouse
* Breed genetically modified (GM) mouse to produce homozygous mice
Common problems in biomedical research
- We think gene X is important for disease Y
- Delete gene X (make a knockout)
- The mice die young, or no viable pups are born at all
- Deleting the gene is embryonic lethal
- To combat this, conditional knockouts have been developed
Conditional knockouts
- Can be conditional around timing, where or when it takes place so researchers can regain some control by using the Cre-lox system
Cre-lox system
- Found in nature within bacteriophages
- Full name is Cre-recombinases (molecular scissors)
- LoxP site (Flox) is recognised by Cre-lox
Conditional or inducible knockout
- Cre activity is controlled in the same way - induced
- The knockout or other modifications is conditional (eg. promoter driven, drug induced)
Tamoxifen
Tamoxifen is used to treat breast cancer and binds to the oestrogen receptor - modified so that it will only activate in the presence of Tamoxifen.
Example
* Conditional deletion of the transcription factor gene SOX9 in adult mice
Hypothesis
* SOX9 is a core regulator of ECM genes during fibrosis. If SOX9 is deleted, no viable pups will be produced; therefore, only delete SOX9 in hepatic sellate cells. However, the problem with this is that we do not know if the particular promoter is active during all stages of development. It is only as specific as the promoter you choose
Conditional or inducible
Methods for controlling when and/or where gene deletion occurs:
* Temporal: in the presence of a drug
* Spatial: only where/when a promoter is active
* Both
Tools needed for targeted gene editing
- Find/search
- Replace
- Delete
- Edit
- Cas9 and transcription factors exist in nature
Protein guided gene editing
Meganucleases
* Endonucleases from microbes
* In nature, they recognise short nucleotide sequences
Zinc finger nucleases
* Artificial restriction enzymes
* Fuse a zinc finger DNA-binding domain to a DNA-cleavage domain
CRISPR
- A bacterial adaptive immune system
- CRISPR stands for clustered regularly interspaced short palindromic repeats
Cas enzyme
- A CRISPR associated endonuclease which is an enzyme that cuts DNA molecules
Natural DNA repair pathways
- CRISPR Cas 9 can be used to make targeted double strand breaks in DNA
- Natural repair processes try to repair these breaks
- We can hijack these repair mechanisms to make targeted genome edits
Non-Homologous End Joining - Efficient
- Error prone
- Requires no template
- Useful for making a knockout
Homology directed repair - More accurate - uses longer structures of homologous sequence to repair DNA
- Can be used to introduce very specific mutations
- Can introduce insertions less than 10bp away from DSB
- Can insert long sequences
Summary - gRNA (guide RNA) - cheap, relatively easy to design. Able to find all the genomic data on the internet
- Cas enzyme makes double strand breaks guided by the gRNA
- Natural repair processes fix the double strand breaks
- Non-homologous end joining (any fix is better than a double strand break otherwise this will lead to apoptosis of the cell)
- Homology directed repair (this bit is nearby and is the same or similar, insert it)
New CRISPR based RNA guided genome editing
Approaches
Base Editors
* Used to make point mutations
* Enzymatically convert bases
* Based on CRISPR
* No strand breaks
Prime Editors
* Prime editing guide RNA
* Specifies the target site and encoded the desired edit
* No double strand breaks
* Used to edit genetic causes of sickle cell in vitro
Why is CRISPR Cas 9 the best gene editor?
The CRISPR Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate and more efficient than other genome editing methods. Since the CRISPR Cas9 system itself is capable of cutting DNA strands, CRISPRs do not need to be paired with separate cleaving enzymes. CRISPR Cas9 was adapted from a naturally occuring genome editing system that bacteria uses as an immune defence. CRISPR Cas9 can be applied directly into an embryo, reducing the time required to modify target genes compared to gene targeting technologies based on the use of embryonic stem cells.
Base editors
Base editing is a novel technology that has the potential to correct certain errors or mutations in the DNA of intact cells. Most pathogenic mutations that cause human disease are single nucleotide polymorphisms (SNPs) that only require a single nucleotide change to correct the mutation.
Ethical concerns
- In war, making biological weapons (humans and crops)
- By editing the human germline, not just the one individual affected
- Germline vs somatic
- Medical vs cosmetic
- Who defines ‘patient’ and who defines ‘normal’
- Don’t know enough about what we are doing
- potentially putting future generations at risk
Successes of gene therapy
- Gene therapy of human severe combined immunodeficiency (SCID-X1) disease
