Gene Editing

Question 1

Reverse genetics meaning and function

Answer 1

Creating mutants to learn the function of gene

Question 2

Describe the three types of mutants and their function

Answer 2

1. Knockout mutants - intentional loss of gene function via alkylating agents such as ethylmethane sulphonate
2. Knockdown mutants - intentional reduction in gene expression by interference RNA - double stranded RNA that binds to mRNA and promoting its breakdown by a protein complex, or siRNA.
3. Over expression - intentional increase in gene expression by insertion of strong promoter/enhancer regions, or disrupting regulatory regions.

Mutations can be induced via insertion mutants created by transposons and T-DNA, or deletions as a results of DS DNA cuts e.g. CRISPR-Cas9

Question 3

What are transposable elements, and transposition.

Answer 3

Transposable elements are sequences that move within the genome, and transposition is the movement of transposons

Question 4

Description of CRISPR-Cas system

Answer 4

1. Clustered regularly interspaced palindromic repeats found in bacterial/archaeal species
2. Made up of two CRISPR repeats with spacer DNA sandwiched between them
3. Spacer DNA originates from viral DNA
4. The spacer + CRISPR repeat forms a complex with Cas9 nuclease
5. The virus with DNA the same as the spacer is called protospacer. When encountered, the bacterial spacer DNA binds to virus antigen and Cas nuclease instigates a double strand cut
6. The Cas nuclease has two domains, the recognition and nuclease domain
7. The DS break leads to repair that creates mutations

Question 5

What is the single guide RNA

Answer 5

The unique sequence that is complementary to target protospacer, contains the spacer DNA

Question 6

Give the main parts of an expression vector to determine the impact of a CRiSPR cas9 region

Answer 6

1. Cas gene
2. sgRNA (single guide RNA)
3. Antibiotic resistance gene (selectable marker)
   4.