Genome editing Flashcards
Zinc finger proteins (ZF)
-small protein domains in which zinc plays a structural role contributing to the stability of the domain
-protein that recognises specific DNA sequence
-Structurally diverse functions
DNA recognition
RNA packaging
Transcriptional activation
Regulation of apoptosis
Protein folding
Assembly and lipid binding
Benefits of ZF nucleases
- Rapid disruption of or integration into any genomic loci
- Mutations made are permanent and heritable
- Works in a variety of mammalian somatic cell types
- Knock out/in cell lines in as little as two months.
How ZF nucleases work
- Design a nucleotide sequence, then put it in the cell by transfection or electroporation
- ZF will recognise the target sequence.
Have control over what genomic sequence you want to target and since you designed it, it will recognise the host genome and bind to it. - Once ZF binds, then have ability for restriction enzyme to cut. Have a protein not DNA bound dsDNA. Cuts DNA
DNA can join by NHEJ or homologous recombination
Two important domains
- DNA binding domain=will recognise the target you are targeting in the genome sequence you are editing
- DNA cleaving domain=recognises restriction enzyme site of FokI
ZF:
Non homologous end joining
Homologous recombination
NHEJ=DNA is broken, the DNA is removed and then the DNA will just rejoin using DNA ligase
Homologous recombination= can insert another gene/exon using homologous recombination
TALENS
Transcription activator like effector nuclease
- Used to modify the genome of any organism
- Can induce mutation (via NHEJ) or insert DNA
- Identify target sequence
- TALEN sequence is engineered
- TALEN in inserted into a plasmid
- DNA transcription to produce mRNA
- mRNA translated to produce the functional TALEN
- TALEN binds and cleaves target sequence
- Introduction of error or new DNA sequence
TALEN example
1yr girl with acute lymphoblastic leukemia
- bone marrow makes to many immature B cells
- > CD19 protein
- bone marrow transplant
- engineered immune cells that can seek and destroy cancer cells without harming the patient
- cancer of the blood, too much protein due to a genetic mutation
TALEN example
Chimeric Antigen Receptor T cells (CAR T-cells)
- Carries an antibody that tracks and kills any cells that make CD19
- CD19 is found on the surface of B cells, type of WBC
- TALENs used to cut a gene in the T cell that produces a protein called T cell receptor alpha chain
- That protein allows T cells to distinguish between a persons own cells and invaders
- Cutting out the gene means the T cells can no longer recognise anything as foreign.
- Stops patients body from rejecting the engineered CAR T-cells
- Then gave patient an antibody drug that kills that patients own T cells, letting the new donor cells grow
- 1 month after treatment, no signs of leukemia
CRISPR
Cluster Regularly Interspaced Short Palindromic Repeats
- Molecular scissors, cutting and replacing DNA letters in an organism’s genome with precision and ease
- RNA guided
- Precision DNA cutting
- Can edit multiple cells
Delivery of CRISPR
- Viral delivery e.g. adenovirus (dsDNA0, AAV (ssDNA), Lentvirus (RNA)
- Lipid nanoparticle delivery
- chemical method
- DNA is coated in lipid, making it easier for it to move across the membrane - Direct nucleic injection e.g. plasmid
Genome editing tools
Can use genome editing tools (ZFN, TAKEN, CRISPR-Cas9) to:
-Gene correction
-transcriptional regulation
-multiplex gene targetting
-gene knock out or report gene insertions
Achieve by homologous recombination and non homologous end joining.
CRISPR Works
- Based on the natural system used by bacteria to protect themselves from viral infections
- When bacterium detects the presence of virus DNA is produces 2 short RNA, 1 of which contains a sequence that matches that of the invading virus.
- These 2 proteins form a complex with a protein called cas9
- Cas9 is a nuclease, an enzyme that can cut DNA
- When the matching sequence (guide RNA) bonds to its target in the viral genome, the cas9 cuts the target DNA disassembling the virus.
Manipulating CRISPR
- Can be engineered to cut not just viral DNA but any DNA sequence at a precisely chosen location by changing the guide RNA to match the target
- If the guide RNA matches, cas9 cuts
- When this happens the cell will try to repair itself which can lead to mutations and the gene being deactivated allowing researchers to understand its function. Mutations are random
- But can be more precise. Can replace mutant gene with a new copy. Can add another piece of DNA that carries a desired sequence
- Once CRISPR system makes a cutm the DNA template can pair up at the cut ends, recombining and replacing the sequence with a new version
- Can be done in stem cells which can give rise to many different cell types and fertilised eggs
- CRISPR can be used to target many genes at once.
