Theme 2 Lecture 8

Question 1

Describe the CRISPR/Cas9 system for genome editing and its key components.

Answer 1

CRISPR/Cas9, a genome editing tool
Uses CRISPR RNA and (TRACrRNA) to guide the Cas9 to specific DNA sites for cleavage.

crRNA (complementary to target DNA),
TracrRNA (forming a guide sequence with crRNA)
Cas9 (RNA-guided DNA endonuclease).
The system requires a Protospacer Adjacent Motif (PAM), typically a ‘NGG’ sequence
PAM is required to cut DNA
Thus Cas9 cleaves DNA upstream of PAM.

Used for precise genomic DNA manipulation.

Question 2

Genome editing outcomes

Answer 2

Three types:
Gene addition homology directed repair
Gene deletion cleavage by two nucleases at the same time
Gene disruptions via small insertions/deletions
In conclusion advanced genome editing allows gene research and creation of disease models

Question 3

CXCR4 receptor(crucial in cancer)

Answer 3

Cuts its sequence at the N-terminus
Homology directed repair adds nanoluciferase into the coding sequence
Thus allowing visualization

Question 4

HiBIT tag

Answer 4

11 amino acid sequence
Thus allowing for efficient tagging to the N terminus of the CXCR4 receptor

Question 5

Conditional KO of mice (Overview of Cre-LoxP) Think of diagram

Answer 5

Cre-LoxP System:
Targets and removes specific genes in certain cells.
Uses “floxed” genes (flanked by LoxP sites) and CRE recombinase.
Function:
CRE recombinase deletes the gene between LoxP sites.
Leads to GFP expression in cells where gene is removed.
Use:
For specific cell types (like endothelial cells) using cell-type-specific promoters.
Example:
Knockout beta adrenergic receptors in endothelial cells: GFP shows where the gene is knocked out.

Question 6

Phage display to get the best antibodies for specific antigens

Answer 6

Gene of interest cloned into gene 3 protein of Phage.

Protein (antibody or peptide) displayed on surface of phage as fusion protein.

Phage library incubated with immobilized antigen

Bound phage eluted and infected into E-coli and amplified.

Enriched population of antibody or peptide fragments for antigen

Question 7

Ranibizumab

Answer 7

Targets VEGF and prevents angiogenesis and there are some other drugs that have all been used via the phage route.

Question 8

Nanobodies-single domain fragments

Answer 8

Camelids (e.g., camels, llamas, alpacas) produce heavy chain antibodies with functional variable regions.

Nanobodies are tiny, efficient antibody parts used for specific antigen targeting in science and medicine

Question 9

Nanobody binding to CXCR4

Answer 9

Nanobody VUN400 effectively binds to the CXCR4 receptor, facilitating luminescence through complementation of HiBiT and LgBiT. This nanobody has applications in studying receptor interactions, even with endogenous receptors.

Question 10

Overview of P2Y12 and P2Y2 Receptors and Their Roles

Answer 10

Activated by ADP=Platelet aggregation
Activated by ATP=Cancer cell metastasis
Used for studying inherited diseases for humans.

Question 11

P2Y1 AND P2Y12 receptors roles

Answer 11

Platelet aggregation
P2Y1 signals
P2Y12 Amplifies which antagonises ADP

Question 12

Why is P2Y12 crucial for thrombosis?

Answer 12

In mice with P2Y12 knocked out in platelets, in vivo bleeding time is significantly prolonged, indicating a crucial role in thrombosis regulation.

Question 13

Platelets(move from blood to tissues) and cancer metastasis

Answer 13

Apyrase reduces ATP and ADP levels, hindering platelet-assisted tumor migration.
Targeting P2Y2 receptor may disrupt cancer metastasis.

Question 14

Mice Lacking Munc 13-4 Protein:

Answer 14

Munc 13-4 protein’s absence reduces tumor cell metastasis by inhibiting ATP release from platelets

Question 15

Conditional KO of P2Y2 in endothelial cells

Answer 15

P2Y2 receptors help endothelial cells retract, aiding tumor metastasis.
Blocking P2Y2 receptors could be a strategy to prevent cancer metastasis.