CRISPR CAS 9

Question 1

What is CRISPR - Cas 9

Answer 1

a genome editing tool which cuts DNA at precise locations in order to
- Delete genes (knock-out)
- fix mutations in genes
- insert new genes (Knock-in)

CRISPR - Cas9 naturally occurs in bacteria essentially the immune system

Question 2

CRISPR-Cas9 in bacteria

Answer 2

Bacteriophages are a type of virus that can infect bacteria by inserting their genetic material into the bacterial in order to hijack the cell and replicate new viruses

CRISPR -Cas9 protects bacteriophages (virus) by making a copy of its DNA and inserting it into the CRISPR sequence

Question 3

CRISPR-Cas9 defence system against viruses

Answer 3

It allows the bacteria to ‘store’ a memory of a viral DNA after infection, ensuring that the next time the same virus invades the DNA will be identified and broken down by a special endonuclease cas-9

Question 4

Exposures:

Answer 4

When a new virus injects its DNA into the bacteria, bacterial enzymes (cas1-cas2 complex) identify a PAM sequnec on the injected DNA and cut a sequence of the DNA. This sequence becomes a new ‘spacer’ in the CRISPR sequence

Question 5

Spacers

Answer 5

The spacers are the different colours they are from different bacteriophages. Unique from the phage that infects the sequence

Question 6

Expression:

Answer 6

• Each spacer sequence is transcribed into an RNA molecule known as guide RNA (gRNA)
• the gRNA forms a CRISPR - Cas9 complex with a Cas9 protein programming the cas-9 protein to target the gRNA sequence
• the target DNA sequence has complementary base pairs to the gRNA

Question 7

Extermination

Answer 7

CRISPR-Cas9 complex scans the bacterial cell for viral DNA that is complementary to the sequence on the gRNA (using PAM to narrow down the search).

Once found, the Cas9 cuts both strands of the virus DNA, creating blunt ends, inactivating the viral DNA

Question 8

CRISPR-Cas9 in gene editing

Answer 8

1) create a synthetic gRNA strand that is complimentary to the target DNA sequence

2) Combine the synthetic gRNA strand with a Cas-9 enzyme to create the CRISPR-Cas-9 complex

3) Inject the CRISPR-Cas9 complex into the target cells

4) CRISPR-Cas9 complex finds the PAM sequence and checks the gRNA sequence against the DNA

5) Cas9 cuts the DNA at a specific sequence

6) the cell to attempt to repair the cut into DNA

7) when repairing DNA the cell may introduce new nucleotides into the DNA
- the cell may introduce random nucleotides, changing the original sequence of the gene, causing it to become inactive (KNOCK-OUT)

• scientist may introduce a new sequence (new gene or allele from the same or different species) into the cell which may be incorporated into the DNA (KNOCK-IN)