5. CRISPR therapies Flashcards
Define genomic editing
Genomic editing - type of genetic engineering in which DNA is inserted / deleted / replaced in the genome of a living organism using engineered nucleases
DNA clevage -> insert target sequence
What are the uses of genome engineering?
Use of genomic engineering:
- gene surgery: genome editing in patients’ cells - in vitro / in situ
- drug development
- animal model creation for research
- genetic variation
- materials
- food
- fuel
What are the types of gene editing that have been used historically?
Engineered DNA binding motifs in enzymes for gene specific editing:
- Zinc fingers: 1990s-2000s, hard to design, hard to synthesise
- TAL effectors: 2010-2014, easy to design, hard to synthesise
- CRISPR: 2013-now, easy to design, easy to synthesise - unlike other use RNA as a DNA recognition sequence - easier to synthesise
How exactly does CRISPR work?
What are the advantages of CRISPR compared with other DNA editing technologies?
- no protein engineering needed
- higher gene editing rates
- large scale experimnets with different nucleases possible
Besides gene editing what else can be achieved with synthetic DNA binding proteins?
Synthetic DNA binding proteins based on their fusion protein to Cas9 can perform:
- gene editing
- transcriptional activation / repression
- Fluorescence
- DNA cooping factors (??)
- Cytidine deamination (epigenetics)
- Reverse transcriptase
What type of DNA cut is performed in CRISPR gene editing?
DNA is cut via double strand break (DSB)
- DSBs in cells naturally recognised as DNA damage - can be highly toxic to cells
-> then use elaborate mechanisms to sense and repair DSBs
What are the DSB repair mechanisms used in gene editing CRISPR?
DNA DSBs repaired by inserting the target sequence - mutagenesis performed by DSB repair via:
- non-homologous end joining (NHEJ)
- homology directed repair (HDR)
Explain NHEJ in DSB repair
Non-homologous end joining (NHEJ) error prone:
1. DSB DNA ends processed by endonucleases -> change ORF - introduce mutations
2. Ends joined
3. Mutation introduced - deletion / insertion of variable length
=> can be used in research to create gene KOs - null -/- mutations - useful for gene KO but not for precise gene editing
Explain HDR in DSB repair
HDR DBS repair - precise sequence correction - rely on a template:
1. DSB DNA ends bound by enzymes directing search for homology between DSB and template sequence
2. Bind the template - synthesise complimentary strand using the template
3. Introduce sequences of the template into the strand
HDR good for precise gene editing / correction using the tenplate sequence
See MOG for good NHEJ, HDR explanations
Are DSB repair mechanisms exclusive?
No, work in equilibrium to repair DSBs - equilibrium between gene KO and gene correction but preferred in different cell cycle phases
Compare and contrast somatic vs germline gene therapy effects
What are the technical challenges of treating genetic disease with gene editing technologies?
Technical challenges of treating genetic disease with gene editing technologies:
- delivering necessary molecules to target cells
- avoiding off-target mutagenesis
- achieving desired genetic change at intended target site
What aspects need to be considered in adressing the challenge of delivering necessary molecules to the right cells in genome ediitng technologies to treat disease?
What are the different approaches ind delivering Cas9 and sgRNA in gene editing? What are their pros and cons
Can be delivered in different forms:
- DNA
- RNA
- Protein
What are the current clinical trials that use CRISPR?
Common theme - in all therapies disease cells are accessible for editing
What is a common approach in CRISPR therapies for treating cell related disease?
Gene editing in appropriate cells is done ex vivo:
- cell environment can be tightly controlled for better effect
- cells with editing outcomes can be selected
- cells with off target mutations can be excluded