Using CRISPR/Cas9 to study neutrophils in Rheumatoid Arthritis (RA)

Question 1

What is the use of a guide RNA (gRNA)?

Answer 1

A guide RNA (gRNA) gives specificity and guides the Cas9 nuclease to the genetic region

Question 2

What is Cas9?

Answer 2

Cas9 is one example of a Cas protein that can cut DNA

Question 3

What do you need for a Cas9 delivery?

Answer 3

Plasmid (circular DNA) with gRNA, Cas9 (and GFP)

Question 4

What are the 6 steps of a CRISPR screen?

Answer 4

1. generate lentiviruses coding for multiple different guide RNAs targeting different genes (one guide RNA/virus)
2. infect cells with a virus preparation titrated so that no more than one virus will enter each cell
3. selection of cells that have been successfully infected by virus
4. a genetically heterogeneous mixed cell population is generated
5. perform screen by separating cells (behaving
   “as expected” or
   not behaving “as expected”)
6. sequence the 20bp guide RNA sequence to identify genes affecting the studied phenotype

Question 5

Why do CRISPR screens?

Answer 5

Understand biology: which genes are important for a certain process
Identify drug targets: mainly be knocking-out targets and see the response

Question 6

What are autoimmune diseases?

Answer 6

Immune cells attacking different parts of the body

Affects around 5% of population

Question 7

What is rheumatoid arthritis (RA)?

Answer 7

Autoimmune disease destroying the joints
Affecting ~75% women
Most common debut at 30-50 years of age
Affects mainly small joints in hand and feet
No cure, just treating symptoms

Question 8

How can a CRISPR screen be useful for rheumatoid arthritis?

Answer 8

Allowing to identify which gene is important for the immune cells (mainly neutrophils) to pass through the joint by knocking it out
Than eventually find a drug that inhibits that gene