Genome editing and sophisticated transgenesis Flashcards
How can we induce mutations in the mouse genome?
- Point mutations - single nucleotide changes
- Insertions into the DNA
- Deletions from the DNA
- Inversions (flipping pieces of DNA)
- Duplications
- Translocations - move DNA
- Expansions
- Rearrangements
- Aneuploidy (generate new chromosomes)
- Copy number variations
- And more…
What might we use modified DNA in mice for?
Study gene function, protein function by inactivating a gene and looking at its effect
Replicating a human genetic defect in the mouse
What is humanisation?
Replace some of the genes of the mouse with equivalent genes from humans
What are the constraints of using mice to model human disease?
Differences in gene expression and genes
Gene dosage can have different effects between human and mouse and we cannot determine the effect for a given gene
Behavioural phenotypes are difficult to model
What is transgenics then?
Transgenesis: Stable insertion of exogenous DNA into host cell’s chromosomal DNA (does not include transient transfection when culturing cells)
What is the earliest model that transgenics is based off?
• Earliest transgenics based on the fact that exogenous DNA added into a nucleus (or even into cytoplasm) sometimes becomes integrated into the cell’s own chromosomal DNA due to:
o probably due to the action of DNA repair enzymes
o usually happens at random (at any chromosomal location)
o but, hot (regions that more likely to take up the DNA) and cold spots (Resistant to taking up DNA) exist
o single or multiple copies of a gene or fragment of DNA
What is homologous recombination?
• If we put a DNA sequence into an organism with the same DNA i.e exogenous DNA with the same sequence as endogenous DNA can lead to exchange: known as homologous recombination
What do genome editing techniques do?
• Genome engineering techniques use modified nucleases to cut (two strands), or nick (one strand) genomic DNA
o endogenous DNA repair enzymes cause mutations or can be directed to insert novel DNA sequences
What are the 3 classical techniques for mouse transgenics?
o Random
o DNA microinjection into zygotic pronucleus
o Relatively quick & easy (you could do hundreds within a morning session)
o We then inject these into a pseudo pregnant female and allow them to develop. They can go to birth or we can do a transient assay by looking at the embryos injected.
o Transient assays possible (we can look at embryos, for example)
o Targeted 1 (target where the mutations occur within the genome) this is the first method:
o Homologous recombination in embryonic stem (ES) cells
o Relatively slow and difficult
o Very powerful and flexible
o Targeted 2 (second method)
o Genome engineering techniques in ES cells or embryos such as crispa
o Ease and speed depend on the technique used
o Possibility of off-target effects (other effects of modifying genome we may not be aware of)
What can we use pronuclear injection for?
- Comparison between a wild human gene and compare to a mutated human gene
- Repress a gene via injection of shRNAi, we can inhibit RNA or degrade it. or block translation
- Express gene with another promoter
- Alter gene expression with Cre recombinase
Describe the process of pronuclear injection
- We super ovulate female mice and mate them
- The fertilised eggs are collected and allowed to mature until the male pronuclei starts to move to the centre of the zygote
- Hold the zygote via suction (microcapillary) and inject DNA into male pronucleus
- Harvest the pups and screen for transgene incorporated.
What must our random transgene contain?
Must consist of a complete transcriptional unit
A promoter - to bind RNA polymerase
Enhancer - direct levels of high gene expression
TS start site
The gene itself with a ATG start site and stop codon
Intron to stop transcription and PolyA to stabilise gene construct
How can we generate a Rosa26 beta-geo transgene?
- We can take a DNA construct that does not contain a promoter but does contain a reporter gene:
Beta gal (beta galactosidase) and neomycin controlled by PGK
Fuse the gene regions to produce Rosa Beta Geo and is only expressed at if it randomly integrates into a gene location of expression. Beta gal expression is now driven by endogenous regulatory regions.
B-geo construct is then injected into ESCs, integrates into DNA where promoter expresses the gene. Then grow these cells in the presence of neomycin and inject into female to acquire the construct we want
Where are ESCs taken from?
Can these cells be genetically manipulated
Inner cells mass cells that are pluripotent and can give rise to to any tissue inside an adult
Yes they can via Homologous recombination
This is homologous recombination -> introduce a gene we want into the embryo
Describe the overall process of homologous recombination
- If we take ES cells that have been maintained in culture of genetically modified and inject those into a host blastocyst
- This blastocyst can be implanted into a foster mother
- The ES cells will integrate randomly to tissues within the organism
- The mice that result from this type of modification are called chimeras because they consist of two cell types (there own and the ES cells).
- These mice can be bred with wild type mice to pass on the gene if it has successfully integrated itself into the genome
What is homologous recombination?
The process of modifying ESCs before we put them into a foster mother, we can insert genes we want to be expressed.
How is homologous recombination performed?
- Here we have a chromosomal region in blue and an identical region of DNA is shown in orange (the homologous DNA)
- DNA repair mechanisms might potentially cause the homologous DNA to be inputted back into the chromosomal region of DNA, so we have the swapping of DNA
- This repair mechanism only recognises the end segments of the DNA so we can input the genome that we want, such as an exogenous piece of DNA into the central part.
- So we can change what we put into a central portion of DNA by making sure the end sequences are the same.
Using homologous recombination, how can we select for our gene construct?
How do we stop random integration of exogenous pieces of DNA we don’t want?
Replace our endogenous DNA with exogenous DNA containing our neomycin resistance gene, expose the cells to neomycin to positively select for cells that have up taken the gene using G418.
HSV-tk is a negative selector and forms a toxic by product when exposed to ganciclovir that kills the cells causing them to die.
So we only get cells with the correct exogenous piece of DNA integrating
Define negative and positive selection
- Positive selection: include a gene in the recombined region that confers resistance of those cells to a toxic drug
- Negative selection: include a gene in the construct that confers sensitivity to a toxic drug in those cells in which random integration has occurred.
Give this a read
Having modified the mouse genome by introducing an additional gene, we may also want to control the timing of gene expression or activity. The following presentation discusses two common ways to achieve inducible gene expression in the mouse.
Give an example of a system that can turn on gene expression
Tet-on/Tet-off system
Summarise the tet-systems in mammals
What 2 transgenes are required in a tet-on/tet-off system?
- The Tet-On® and Tet-Off® systems. Requires 2 transgenes, one making a drug responsive repressor (or activator) protein, the other has a response element with the gene of interest.
- A. Tet-On® System. Four amino acid changes to TetR (tet repressor) alter its binding characteristics and create reverse TetR (rtTA (the activator)), which binds the TRE (tet response element) in the presence of doxycycline and activates transcription.
- B. Tet-Off® System. The tet-controlled transcriptional activator (tTA) is a fusion of the wild-type Tet repressor (TetR) to the VP16 activation domain (AD) of herpes simplex virus. tTA binds the Tet-responsive element (TRE) and activates transcription in the absence of tetracycline or doxycycline.
Describe the bacterial Tet-on/Tet-off system
- The Tet-repressor (TetR) will bind to the Tet-response element and turn off gene expression in the absence of tetracyclin
- When tetracyclin is present it binds to TetR activating gene expression, turning on genes that allow the bacteria to survive
Describe the modified mammalian Tet-on/Tet-off system
TetR is modified where the repression domain is replaced by a trans activator domain from VP15 (a viral protein from HSV) called tTA
This tTA protein binds to the TRE and turns ON gene transcription in the absence of Tetracycline
Adding tetracycline prevents binding of tTA and turns off gene expression
tTA can be modified by a change of 4 amino acids that alters its binding specificity now called reverse tTA (rtTA)
In the absence of tetracycline it cannot bind to the Tet response element to gene activation is turned off
Add tetracycline it changes the binding specificity of rtTA protein to it binds to the tet response element that drives transcription
How can the oestrogen receptor pathway be altered to generate other oestrogen responsive proteins?
In a cell not receiving a oestrogen signal the oestrogen receptor is held in the cytoplasm by HSP90 which keeps oestrogen receptor protein in cytoplasm
If the cell encounters oestrogen it binds to the oestrogen receptor in the cell cytoplasm, HSP90 dissociates
Oestrogen receptor can translocate into the nucleus, dimerise with other oestrogen receptors to drive transcription of downstream target genes
The oestrogen receptor binding domain can be altered and fused to a strong transactivator
In the presence of oestrogen, the modified oestrogen receptor will trans activate anything downstream of an oestrogen response element
LBD is further modified so it has a higher sensitivity for an oestrogen antagonist called Tamoxifen so not activated by oestrogen
What are knockout mice?
-The creation of a null (completely non-functioning) mutation for a specific gene
What are knockout mice sometimes used to model?
-Sometimes used to model a human loss of function mutation/disease
How are knockout mice usually made?
-Usually done by removing all coding exons, or essential exons near the 5’ end of the gene
How are mouse models of cystic fibrosis created?
- Cystic fibrosis caused by an inactivating mutation of the CTFR by insertion of exogenous gene sequences
- CTFR delta F508 mutation introduced by homologous recombination
-CTFR null mice have a more severe phenotype than deltaF508 mutations
What happens to the mouse mutants of cystic fibrosis and due to what?
Both mutants die shortly after birth
-But from intestinal obstruction, not lung problems
What is Cre?
Cre is a site-specific recombinase
What is loxP?
34bp recognition site of Cre
What does the effect of cre-loxP recombination depend on?
-Effect depends on the orientation of the loxP sites
What happens if the 2 loxP sites are placed in the same orientation on the same piece of DNA?
- Most commonly, the loxP sites are placed in the same orientation leading to a recombination of the 2 sties
- The recombination will lead to the removal of the intervening sequence between the loxP sites
What does adding cre recombinase to 2 loxP sites present on 2 different sites of DNA in the cre-loxP system lead to?
-Adding Cre recombinase will lead to crossing over of sequences
What does adding cre recombinase to 2 loxP sites present on the same piece of DNA in the cre-loxP system lead to?
-Adding Cre recombinase will cause recombination between the 2 loxP sites and removal of intervening sequence
What is a conditional allele referred to as?
Conditional allele which is referred to as a floxed gene
How are loxP sites inserted into DNA?
How do we delete a gene?
- To insert loxP sites, the region of genomic DNA is cloned into bacteria and modified, then used to generate targeted embryonic stem cells by homologous recombination
- Targeted ES cells are used to generate mouse line with a floxed allele
- These mice are normal
- Then those floxed mice are crossed with a Cre mouse and that causes recombination between the 2 loxP sites in the same orientation and that’s now the deleted gene
What are the 3 ways that expression of Cre can be driven?
• eg knocked in to an existing gene, so controlled by that gene’s promoter
o OR
• driven by a promoter of its own – this could be expressed in all tissues, or some and expressed all the time, or at specific times
o OR
• can be turned on/off by specific drugs that are given to the mouse eg tamoxifen
Summarise How a gene is therefore knocked out
Generate a floxed mice -piece of DNA we want to deleted flanking LoxP sites
Generate a Cre-recombinase mouse being expressed
Cross mice together
Offspring have the deletion in a specific location
How is Cre made conditional?
Fusion of Cre recombinase to oestrogen receptor ligand binding domain (ER^T) = Cre-ER^T. This has a high affinity for tamoxifen
Cre-ER^T is held in cytoplasm by HSP90
Expose to Tamoxifen to dissociate HSP90
Cre-ER^T causes recombination between LoxP sites
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How is genome editing different to transgenesis?
- The vast majority of models are mouse models, and until recently, all made by transgenesis – that is the introduction into the mouse genome of “unnatural” DNA, artificial or from another species
- Recently, genome editing technology has allowed changes to be made directly to genomic DNA sequences (without adding extra sequences)
What are the 3 broad classes of genome editing nucleases?
- Zinc finger nucleases
- TALENs
3.CRISPR
What is the zinc finger nuclease?
A protein consisting of a DNA-cutting enzyme and a DNA-grabbing region that can be tailored to recognise different genes
What are TALENs?
A protein containing a DNA-cutting enzyme and a DNA-grabbing region that can be programmed to recognise different genes, but it is easier to design than zinc finger nucleases
What is CRISPR?
A DNA-cutting protein guided by an RNA molecule that is able to find the specific gene of interest
How do zinc finger proteins work?
- Need to be individually designed to recognise a specific DNA sequence within the genome
- They bind to that sequence and cut it
How do TALENs work?
-Have to be designed for a specific DNA sequence
How does CRISPR work?
- Has specificity that is conferred by the guide RNA
- The nuclease which cuts the DNA is Cas9 and will work on any sequence of guide RNA as long as trace RNA remains there
What does CRISPR stand for?
-CRISPR stands for clustered interspersed short palindromic repeats
What is CRISPR-Cas9 based on?
-Based on bacterial adaptive immune system
What can CRISPR-Cas9 be used for?
-Can be used for a wide variety of species and cells in culture
What does CRISPR-Cas9 require?
-Requires a guide RNA with a protospacer adjacent Motif
Where do genome edititng techniques occur
Pronuclear stage of embryos - nucleases injected
Steps involved in the CRISPR-Cas9 system
- We’d make a single guide RNA (sgRNA) which consists of a target specific crRNA sequence attached to a tracrRNA
- This sgRNA will be added into a Cas9 protein
- This will then induce the double stranded cleavage of the genomic DNA at a specific location
- Then the cellular machinery will try repair the double stranded break by non homologous end joining(NHEJ) or homology-directed repair(HDR)
- Imprecise NHEJ mediated repair can produce insertion and/or deletion mutations of variable length at the site of the double stranded break
- HDR mediated repair can introduce precise point mutations or insertions from a single stranded or double stranded DNA donor template
Why are there CRISPR variations?
CRISPR-Cas9 has off target effects
What is an example of a CRISPR variation method?
Genome engineering by double nicking with paired Cas9 nickases
How does Genome engineering by double nicking with paired Cas9 nickases work?
What can Crispr Cas9 also be used as?
- Cas9 has been modified so it has nickase activity
- This means that it cleaves only one strand of genomic DNA and not both
-By targeting 2 DNA nearby, you can have 2 specific events and can then use HDR
Transcriptional activator
repressor
reporter gene
