hl mutation and gene editing

Question 1

Gene knockout as a technique for investigating the function of a gene by changing it to make it inoperative
- Students are not required to know details of techniques.
- Students should appreciate that a library of knockout organisms is available for some species used as models in research.

Answer 1

• DNA is prepared with a base sequence that allows it to be inserted into the genome of embryonic mouse cells as a replacement for a target gene, which is therefore deleted.
• To find out the function of a gene or its base sequence, targeted knockout of genes by deletions or insertions are carried out.
• Gene knockout is often carried out in mice as model organism. The phenotype of the mice containing the engineered genes is then investigated to find out which traits have been changed by deletion of the target gene. A whole library of knock-out strains can be generated that way.

Question 2

Use of the CRISPR sequences and the enzyme Cas9 in gene editing
- Students are not required to know the role of the CRISPR–Cas system in prokaryotes. However, students should be familiar with an example of the successful use of this technology.

Answer 2

• CRISPR-Cas9 is gene editing based on a natural system that exists in many species of prokaryotes, which use it as a defense against viral attacks.
• In nature, the main two elements for this system are:

CRISPR regions within the genome
The enzyme Cas9

Question 3

What does CRISPR sequence stand for?

Answer 3

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• The spacers are effectively viral DNA which has been cut previously, and then inserted into the bacterial DNA as “memory” for future infections.
• When bacteria are infected by a virus, they first use their CRISPR system within their genome to cut up the invading DNA and insert pieces (spacers) of it into their own genome as a memory of the infection. Two sequences within the bacterial CRISPR system are then transcribed into RNA:

Question 4

Hypotheses to account for conserved or highly conserved sequences in genes
- Conserved sequences are identical or similar across a species or a group of species; highly conserved sequences are identical or similar over long periods of evolution.
- Two hypotheses for the mechanism are functional requirements for the gene products and slower rates of mutation.