D1.3

What are SNPs

• single nucleotide polymorphisms

• they are base substitution mutations (only 1 nucleotide is replaced)

• can be 2 types:

  • synonymous substitution (neutral, doesn’t change AA sequence)

  • non synonymous (changes AA sequence, protein malfunction)

What are the 2 types of mutagens?

• chemical (mustard gas, nitrous acid, formaldehyde)

• radiation (UV, can lead to DNA damage, like single or double strand breaks)

  • single strand breaks: impede movement of replication fork and thus stops continuity, resulting in errors or complete stop

  • double strand breaks: more severe, can completely halt replication

When would there be a higher risk of mutation

• when cells that are actively undergoing cell division are exposed to mutagens

Which N bases have a higher probability of mutating than others? why?

• cytosine —> can undergo deamination (a spontaneous chemical mutation), converting it into uracil

• due to chemical properties and vulnurability

What are consequences of mutation in germ vs. somatic cells

germ

• the only way mutation can be passed down

• can alter chromosome number or gamete gene sequence

somatic

• can cause diseases

• not passed into offspring

Compare and contrast neutral vs. silent mutation

Neutral: occurs in non-coding regions

Silent: occurs in coding sequence but doesn’t alter AA sequence due to degeneracy of code

What is gene knockout technique?

• a specific gene is intentionally removed or changed so the expression is permanently prevented

What are the 2 components in CRISPR-Cas9

• CRISPR: specific DNA regions in bacteria that have short repeated sequences and unique spacer sequences that are incorporated from foreign DNA encountered by bacteria

• Cas9 (endonuclease) enzyme: cuts DNA at specific target sites on chromosome

Where is the CRISPR-Cas9 system naturally found? why?

• in bacteria as a self defence mechanism against invading foreign DNA

• helps record short sequences of foreign DNA into their own genome (spacers)

• bacteria keeps a record of previous infections to destroy similar one in the future

What are the 3 steps in CRISPR-CAS9

1. when foreign DNA is found to match a CRISPR spacer, a corresponding crRNA identifies it and binds to viral sequences

2. this tells cas9 to the target DNA to make cuts in it

3. results in a double stranded break that can be repaired

What is the main use of CRISPR-Cas9?

• for making sgRNA (Single guide RNAs) to target specific genes for modification

• sgRNA —> designed to target and bind to DNA sequence

  • guides Cas9 to the location to cut DNA, where ds breaks

  • after break is made, can be modified

list 4 applications of CRISPR-CAS9

• gene therapy

• agriculture

• disease modelling

• genetic engineering

Describe the 10 steps for CRISPR-CAS9 gene editing tool and the 3 main components

1. there is a DNA double helix with mutation

2. guide RNA is made so it matches mutated DNA

3. Cas9 is added to gRNA mix

4. cells are injected with gRNA + Cas9

5. gRNA identifies the mutated DNA

6. Cas9 requires a PAM (protospacer adjacent motif, which is a few nucleotides long) to attach to the target DNA

7. Cas9 cuts out mutated sequence

8. this results in a double strand break

9. faulty gene can be replaced by correct DNA or a new gene

10. the cell will attempt to repair the break, which silences the targeted gene

What is a guide RNA?

it can be generated in lab to match a target sequence

it acts as a guide to show the enzyme where to cut