L6: Gene editing Flashcards
(32 cards)
Define homozygous
Same Alleles.
This refers to an organism having two identical alleles for a specific gene. For example, if an organism has two copies of a knockout allele (KK) or two copies of a wild-type allele (WW), it is homozygous for that gene.
Define: heterozygous
Different alleles.
This refers to an organism having two different alleles for a specific gene. For instance, if an organism has one knockout allele and one wild-type allele (KW), it is heterozygous for that gene.
In sickle cell disease, which codon is replaced?
Adenine replaced by thymine
In sickle cell disease, what amino acid is made by the substitution of Adenine to thymine and from what to what?
from glutamic acid to valine.
Explain how guide RNA is designed for CRISPR-CAS9
Humans share approximately 99.9% of their genomes, and there are extensive DNA databases containing many gene sequences. To design guide RNA (gRNA) for CRISPR-Cas9, scientists can utilize bioinformatics tools and software to identify target sequences next to a protospacer adjacent motif (PAM).
Once the desired gRNA sequence is identified, researchers can either purchase pre-made gRNA from companies like Synthego or design a DNA template that encodes the gRNA sequence. This design is generated using computer software.
After acquiring the DNA template, it can be synthesized by a commercial company. Following that, in vitro transcription using RNA polymerase allows the synthesis of the guide RNA based on the DNA template. This synthesized gRNA can then be used in conjunction with the Cas9 protein for gene editing.
After CRISPR-cas9 has copied and pasted the right sequence into the genome, what are the two methods if can use to join the ends of the double strand breaks back together?
- Non-homologous End Joining
This is the most common method as it is part of the cells innate repair processes.
If the ends are uneven then, the exonuclease can trim them to make them suitable for joining.
The broken strands are brought back together and ligated by DNA ligase.
However, This process can introduce indels (insertions/deletions):
- Small insertions (i.e., free floating nucleotides)
- deletions due to the imprecise nature of the repair, which can disrupt the function of the target gene.
- Homologous recombination
This method is used for precise edits (I.e., knock-ins or specific mutations).
The scientist will need to create a template DNA with the sequence they want to add or change. (can synthesise or buy).
This template will have DNA sequences on either side of it that matches the DSBs.
The single-stranded part of the donor template finds and binds to one of the cut DNA strands at the break. It pairs up with the matching sequences.
Enzymes (DNA polymerases) then add new DNA bases to extend the matched strand using the donor template as a guide.
The newly made DNA gets connected to the other broken strand, often with the help of another enzyme (DNA ligase) to finalise the repair.
This process allows for a precise change or correction in the DNA at the site of the break, enabling targeted gene editing.
What is transfection in gene editing?
When DNA or RNA are artificially added to a eukaryotic cell.
Side quest: this is a key process in CRISPR-cas9/Gene editing.
Name the process in which an electrical current is used to make temporary pores in the phospholipid bilayer, allowing scientists to insert DNA or RNA into a cell.
Electroporation
What is the primary purpose of gene editing
Analyse cellular and molecular interactions and gain insights that could have therapeutic benefits for disorders and diseases, and facilitate scientific discovery. This includes gene functions
How do dsDNA breaks facilitate gene editing?
NHEJ and HRR and their respective endo/exonucleases help join the DNA that has been added so it integrates into the host DNA.
What are insertions and deletions (Indels)
In NHEJ - when the exonuclease trims the ends of the dsDNA base, it can trim off a few nucleotides (deletion), or free floating nucleotides can be added before the repair is complete.
In HRR it is the same. additionally,Insertions can also be added if the template DNA has mismatches or sequence variations.
What major challenge is associated with gene editing?
Limiting off-target effects and achieving specificity.
What is a site-specific nucleases?
An engineered protein that introduces DSBs at precise locations in the genome. CRISPR, ZFNs and TALENs are examples of methodologies that use site specific nucleases alongside other enzymes such as CAS9 for CRISPR and FokI for ZFNs and Talens to create complexes that find and cut DNA at desired loci.
What are meganucleases?
They are a DNA cutting enzyme, and a subtype of endonuclease. They are engineered and have the ability to cleave long specific DNA sequences (~20-30bp.) They are used in TALENs and other biotechnological applications.
How do Zinc Finger Nucleases operate?
They are engineered proteins.
They consist of a DNA binding domain made of Zinc finger motifs.
The motifs recognise 3 base pairs.
They include the FokI nuclease domain for cleaving DNA.
What is the significance of a dsDNA break in gene editing?
Scientists can leverage repair mechanisms.
This allows for targeted modifications to genes.
How do TALENs differ from ZFNs
TALENs use building blocks that can recognise individual nucleotides which makes them simpler to create and customise for specific DNA targets.
What is Non-Homologous end joining?
It is a repair mechanism in DNA.
Uses exonuclease to trim the ends of DNA following a DSB.
What is homologous recombination?
It is a DNA repair pathway.
It uses a homologous template strand, usually from a sister chromatic to accurately repair the DSB.
The process involves endonuclease’s that create the DSB and mechanisms like D-loops for stabilisation and Holliday junctions to facilitate repair.
Why are targeted DSBs required for efficient gene editing?
To make sure the repair mechanisms can be directed to specific gene loci.
What is the average size of a unique target sequence in the human genome?
~20-25 nucleotides.
Why is specificity important in gene editing?
It limits off-target effects.
What is a key feature of the ideal engineered nuclease?
It should allow for precise targeting with minimal off-target cleavage.
What role did positive-negative selection play in early gene targeting?
It helped identify successfully/unsuccessfully modified cells in experiments.
