Cut it out, from knockouts to gene editing and therapies Flashcards
2 Shortcomings for CRISPR approach
- The genetic abnormalities that arise in human individuals are not always clean. For example, small chromosomal abnormalities, which are thought to give rise to conditions such as autism and schizophrenia, often encompass more than one gene. This can complicate the interpretation of experiments performed using IPS cells derived from such individuals.
- Normal genetic variability between human subjects means that cell lines need to be made from multiple individuals, with both a particular disease of interest and unaffected individuals, to have the power to detect subtle cellular differences that could be involved in the disease process.
What is the solution for the problems mentioned?
By engineering known disease-causing mutations into an IPS cell from an unaffected individual. This allows a precise genetic change to be made, and the original cell line can be used as what is known as an isogenic control.
In other words, the control cell line that you are comparing the mutated cell line to is genetically identical, except for the mutation of interest. This means that any differences observed can be attributed with confidence to the mutation, and cannot be arising, or indeed masked by, some other differences in the genetic background.
Whats is CRISPR?
CRISPR stands for clustered regularly interspaced short palindromic repeats.
This name refers to the DNA sequences discovered in the genomes of bacteria. These unusual DNA sequences were found to be part of a type of innate immunity that bacteria have developed against viruses. The CRISPR sequences in the bacterial genome arise because when the bacterial cell is infected by a virus, the viral DNA is processed by the bacteria into short segments and integrated into the host bacteria’s genome. The bacteria then use these sequences of viral DNA to guide an enzyme, called a nuclease to destroy any subsequent viral infections. This is highly specific because the guide sequence, having been derived from a previous virus, is perfectly complimentary to that sequence in the subsequent invading virus. Upon binding to the complimentary sequence in the viral DNA, the attached nuclease introduces a double-strand break in the DNA in activating the virus. Researchers quickly realised the potential of the system as a tool to perform precise genome editing, and the CRISPR system was rapidly adopted by researchers for use in human cells
What is nuclease and how it’s used?
It is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids.
The most commonly used nuclease in research is from the bacterium streptococcus, and is called Cas9. This nuclease typically cuts the DNA within the guide sequence, four nucleotides away from what is known as the protospacer adjacent motif, or PAM sequence. The PAM sequence for streptococcus Cas9 is NGG, where N stands for any nucleotide, and follows on directly after the guide sequence. The PAM sequence is essential for binding of the CRISPR Cas9, but does not form parts of the guide sequence itself.
What are the 2 types of genome editting?
Genome editing (also called gene editing) is a group of technologies that give scientists the ability to change an organism’s DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome
- inactivating the function of a gene
- altering a single base pair to make a point mutation.
What are the two different repair pathways in the cell for fixing double-strand breaks in DNA?
- Non-homologous end joining, is both the quickest and the most commonly used repair pathway in most cells, including pluripotent stem cells.
- Homology-directed repair, so called because it uses a sister chromatid as a template to create a very accurate repair to the break.
Non-homologous end joining is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair, which requires a homologous sequence to guide repair
How non-homologous ways is problematic?
When a double-strand break is repaired by a non-homologous end joining, small sequences of DNA are sometimes added or removed to facilitate the repair process. This change in the DNA sequence of the gene, and therefore the RNA transcript it produces, can be recognised by the cell as abnormal, and the transcripts are degraded before they can be translated into protein. This process of degrading abnormal RNA transcripts is called nonsense-mediated decay, and if both alleles of a gene are mutated in this way, the gene will be knocked out, in other words, unable to make a functional protein
What is amyotrophic lateral sclerosis (ALS) ?
A neurodegenerative disease characterised by the loss of motor neurons and progressive muscle wasting, with death typically occurring within three to five years of symptom onset.
