4B: CRISPR-Cas9

Question 1

Virus

Answer 1

A non-cellular, infectious agent composed of genetic material enclosed in a protein coat that requires a host cell to multiply.

Question 2

Bacteriophage

Answer 2

A virus that infects prokaryotic organisms.

Question 3

Deleterious mutation

Answer 3

A change in DNA that negatively affects and individual.

Question 4

Gene therapy

Answer 4

Repairing genetic mutations by replacing the defective gene with a healthy one.

Question 5

Gene knockout

Answer 5

A technique in gene editing where scientists prevent the expression of a target gene to understand its function in an organism.

Question 6

Applications of CRISPR-Cas9

Answer 6

Research:
-Attaching a fluorescent protein to Cas9 to locate a specific gene in the genome

Dealing with diseases:
-Replacing a deleterious allele with a healthy one
-Modifying cancer-promoting genes to make them less influential

Agriculture:
-Introducing pest and herbicide resistance genes to increase the yield of crop
-Altering genes to promote increased growth rates to improve the yield of crop

Question 7

Steps in CRISPR-Cas9 defence

Answer 7

Step 1: Exposure
Step 2: Expression
Step 3: Termination

Question 8

Exposure

Answer 8

• When the bacteriophage injects its DNA into the bacterium. the bacteria identifies the viral DNA is foreign.
• Cas 1 and 2 are CRISPR associate enzymes that cut out a section of the viral DNA known as the protospacer.
• This protospacer can then be introduced into the bacterium’s CRISPR gene to become a spacer.

Question 9

Expression

Answer 9

• The CRISPR spacers are transcribed along with half a palindrome from the repeat either side of it, and is converted into a RNA molecule known as single guide RNA (sgRNA).
• sgRNA binds to Cas9 to create a CRISPR-Cas9 complex which is directed to any viral DNA inside the cell that is complementary to the sgRNA.
• sgRNA forms a hairpin loop-like structure from the transcribed palindromic repeats either side of the spacer.

Question 10

Termination

Answer 10

• The CRISPR-Cas9 complex scans the cell for invading bacteriophage DNA that is complementary to the one on the gRNA.
• When it is detected, Cas9 cleaves the phosphate-sugar backbone to deactivate the virus.
• Cas9 contains two active sites to cut both strands of DNA and create blunt ends.

Question 11

Cas9:

Answer 11

an endonuclease which can cut at different sequences

• Unlike normal enzymes, it is not specific

Question 12

CRISPR-Cas9:

Answer 12

a complex formed between sgRNA and Cas9 which can cut a target sequence of DNA.

• Bacteria use this complex for protection from viruses and scientist have used it to edit genomes

Question 13

Spacer:

Answer 13

a short sequence of DNA obtained from invading bacteriophages that are added into the CRISPR sequence

Question 14

Protospacer:

Answer 14

a short sequence of DNA extracted from a bacteriophage by Cas1 and Cas2 which has yet to be incorporated into the CRISPR gene

• Becomes a spacer when added into the cRNA

Question 15

Protospacer adjacent motif (PAM):

Answer 15

a sequence of 2-6 nucleotide that is found immediately and next to the DNA targeted by Cas9
- Prevents the Cas9 from accidently cutting through the cRNA sequence with all the genetic material
- PAM code = NGG(N = any nucleotide base)
- Acts as a initiation signal for Cas9(Cas9 looks for the PAM not for a match of viral genetical material)

Question 16

Single Guide RNA(sgRNA):

Answer 16

RNA which is single stranded and has a specific sequence determined by CRISPR to guide Cas9 to a specific site

Question 17

What happens when Viral DNA is cut up:

Answer 17

• Enzymes within the bacterium will naturally act to repair it however the repair mechanisms are prone to errors and which can result in nucleotide additions, deletions or insertions
  • This is advantageous in the case of bacteriophage infiltration as mutations tend to render viral genes non-functional
  • If a mutation does not occur, the sgRNA will find the gene and repeat the process again

Question 18

How to use CRISPR-Cas9 for gene editing

Answer 18

1. Synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA that scientists wish to cut
   2. A Cas9 enzyme is obtained with an appropriate target PAM sequence
   3. Cas9 and sgRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex
   4. The sgRNA-Cas9 mixture is then injected into a specific cell, such as a zygote
   5. The Cas9 finds the target PAM sequence and checks whether the sgRNA aligns with the DNA
   6. Cas9 cuts the selected sequence of DNA
   7. The DNA has a blunt end cut that the cell will attempt to repair
   8. When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site. Scientists may also inject new nucleotide sequences into the cell with the hope that it will ligate together