Gene Editng Techniques Flashcards
What is a genome?
Genetic material that defines an organism. It is the complete set of nucleic acid sequences encoded as DNA within 23 chromosomes.
What was the Human Genome Project?
10 year project in which scientists around the globe sequences the whole human genome.
They identified all human genes, and the aim was to identify what each gene did and what they were used for in the body.
However we still dont now the extact function of each gene and how we can modify all of them for therapy.
________ genes from the 25,000 genes identified in the human genome have been linked to disease.
3,000
- however as cheaper and better sequencing tech is developed this number is expected to increase.
Give some examples of some diseases with genetic links that have been established , and some that are being established.
- sickle cell anaemia, haemophilia
- schizofrenia, hypertension
Why is it difficult to treat genetic diseases?
- many drugs don’t target the “wrong” DNA code which leads to the production of the incorrect protein and thus genetic disease.
- many of these drugs tend to work on the downstream issues ie what has occurred as a result of that genetic disease.
There has thus been a need for new reagents that modify genes.
What is gene editing?
A process in whic h engineered nucleases are inserted in order to replace or remove or insert DNA from a genome.
What is a nucleases?
AN enzyme that can cleave the phosphodiester bonds within a nucleic acid (ie DNA and RNA), allowing for genetic modification. 🧬
Which 3 gene editing techniques have been developed?
- nb they all use different types of nucleases…
- ZFN - zinc finger nucleases
- TALENS - transcription activator like effector nucleases.
- CRISPR/Cas9 - clustered regulatory interspaced short palindromic repeat (CRSIPR)/ Cas - based RNA guided DNA endonucleases (CRISPR/Cas9)
What do we need to create in order to edit the gene
A double strand break at the nucleases target site, by either ZFN, TALENS or crispr cas 9
What happens to the DNA strand after the DNA strand has been broken by TALENs/ ZFN/ Crispr cas 9.
The DNA is repaired by the cells own repair mechanisms, HDR or NHEJ, leading to either deletions, insertions, replacements or corrections of the DNA.
What are the two outcomes for Homologous Directed Repar.
This is when a DNA template is used to find the DSB at sites prior to and post DBA area so that when new bases are inserted, they know exactly where to be inserted.
- gene insertion - addition of a new gene
- gene correction - addition of a correct version of the gene
What is the outcome of Non - Homologus end - joining as a DNA DSB repair mechanism?
The DSB is repaired without the addition of a new gene in the middle.
This is optimal for a disease where there are extra base pairs leading to damage and sub therapeutic effects.
- leads to gene disruption
How do zinc fingers cause DNA DSBs?
Each Zinc finger will recognise a set of 3 base pairs in the DNA , which is where it will make the DSB.
It has a DNA binding domain and a DNA cleaving domain.
The DSBs are made in the DNA at user specified locations.
How do TALENS cause double strand breaks?
Each TLN will recognise ONE base pair unit in a 1:1 ratio - this method is more flexible in the sense that we can design it to find which sequence we want it to cut and thus generate the double strand break.
It too has a dna binding and dna cleaving domain.
Briefly explain how ZFNS and TLNS can be used to create DSBs in order to edit genes in therapy?
In the presence of a faulty gene, we can engineer ZFNS and TLNS to recognise these faults and then design them so that they make double strand breaks at that point.
This will damage the sequence and elicit intrinsic cell repair mechanisms to repair the cell DNA.
These methods were good until CRISPR/cas - 9 came along.
