Lecture 3 - CRISPR III

Question 1

Why is the ability to modify individual bases important?

(4 Points)

Answer 1

Point mutations - implicated in various human diseases due to changes in:
1. Crucial Amino acids
2. Promoter/Operator Sequences
3. Regulatory RNA Sequences

Question 2

(i) What are Cytosine Deaminases?

(ii) Give Two Examples of processes they are involved in?

Answer 2

(i) Base Editor Enzymes, which convert cytosine (C-G) to Uracil (T-A) under specific conditions

(ii) Involved in:
* ApoBEC1 - truncates protein, helping to control lipid metabolism
* AID - class switch recombination for antibody formation

Question 3

Describe the Composition of a BER “Editor tool”

Answer 3

• Consists of:
  1. nCas9 - avoids dsDNA breaks
  2. Cytosine Deaminase (fused to nCas9)
  3. Uracil Glycolase Inhibitor - prevents removal of Uracil to produce Abasic (AP) sites

Question 4

Define CRISPRi (CRISPR Interference)

(3 Points)

Answer 4

Targeting of dCas9 arrays to the coding region of a gene in order to:

1. CRISPRa - activate expression of a gene (e.g., dCas9 is fused to transcriptional activator)
2. CRISPRi - repress expression of a gene (e.g., dCas9 is fused to transcriptional repressor)

Question 5

How can dCas9 enzymes themselves produce inhibitory effect on gene expression?

Answer 5

dCas9 enzymes can bind along coding region of target gene and form R-loops that inhibit transcription of the gene (R-loop - very stable)

Question 6

State Two Examples of how CRISPRi can be utilised in research

Answer 6

1. CRISPRi Phenotype screening
2. Synthetic Lethality

Question 7

Define Transposons

Answer 7

MGEs that move around DNA fragments by excision out/integration into chromosomes via their Terminal Inverted Repeats (TIRs or LE/RE)

Question 8

How do Transposons excise out/integrate into different sites in the genome?

Answer 8

Encode an enzyme (Transposase) that recognises TIRs and cuts/inserts the gene at different sites

Question 9

How do CRISPR Associated Transposons (CASTs) integrate their DNA?

Answer 9

Large DNA Payload (10kb+) is recruited by a cascade interference complex, which uses a gRNA to direct transposon intergration into target site in genome

Question 10

(i) What are Casposases?
(ii) What is their Function?

Answer 10

(i) Gene encoded within class of archaeal transposon that encodes a protein resembling Cas1

(ii) Form tetrameric complex, which integrates DNA into the Archaeal genome in mechanism similar to CRISPR adaptation

Question 11

Compare the targeting of CRISPR Adaptation to that used by Archeael Casposases

(2 Points)

Answer 11

• CRISPR Adaptation - displays preference for PAMs
• Casposases - use ITRs to perform a similar function

Question 12

Describe the Cas2 Recruitment Hypothesis

Answer 12

Suggests that Mutation(s) in the monomer-monomer interface of Cas1 prevented tetramerisation, therefore Cas2 was recruited to connect the dimers