L12: Genome editing using CRISPR/Cas9

Question 1

Define genome editing

Answer 1

A type of genetic egineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucelases

Question 2

What is the CRISPR/Cas system?

Answer 2

It’s a prokaryotic immune system

Question 3

What is the use of XRISPR associated proteins (cas)

Answer 3

They use the CRISPR spacers to recognise and cut exogenous genetic elements

Question 4

How prevalent are CRISPRs in prokaryotes?

Answer 4

They are found in ~40% of sequenced bacterial genomes and 90% of sequenced archae

Question 5

Explain a ‘nick’

Answer 5

This is a single stranded break (ssb) and can be repaired by ligase

Question 6

Name 2 DNA repair mechanisms used for double stranded breaks

Answer 6

Non homologous end joining (NHEJ) and Homology directed repair (HDR)

Question 7

How is NHEJ performed?

Answer 7

DNA is capped by ku/ DNA PKc complex and DNA ligase then the DNA break is fixed.
- Ends are directly ligated without the need for a homologous template!

Question 8

NHEJ is error prone, why and what can this lead to?

Answer 8

As it only aims to fix break, this can lead to ripping out bps, inserting randoms bps or just using any bps to fill the gap
- can cause insertion or deletion of bps

Question 9

How can NHEJ be used to created KO genes?

Answer 9

Either by loss of coding DNA or shifting translational codon reading frame

Question 10

How is HDR performed?

Answer 10

The unbroken strand is used as a template and ATM/ RDA/ RAD51 complex is used for strand invasion and copying to repair the break

Question 11

Which mechanism is preferred for DNA repair and why?

Answer 11

HDR as while it is slower than NHEJ, it is less error prone and is also essential for prevnting cancer

Question 12

Explain how CRISPR can be used as a genome editing tool to create KOs

Answer 12

1. Introduce guideRNA (gRNA) into cell which contains scaffold and spacer
2. gRNA forms complex with Cas9→ leads Cas9 to complimentary sequence and causes a dsb leading to NHEJ or HDR
3. PAM (protospacer adjacent motif) is required for Cas9 to work, joining onto the spacer
4. Resulting in DNA fixing itself, an insertion, deletion or frameshift mutation

Question 13

Cas9N can create nicks. True or false?

Answer 13

True

Question 14

Explain how CRISPR can be used as a genome editing tool to create KIs

Answer 14

1. Introduce guideRNA (gRNA) into cell which contains scaffold and spacer
2. gRNA forms complex with Cas9 but this time we add our own template
3. PAM (protospacer adjacent motif) is required for Cas9 to work, joining onto the spacer
4. Cell uses this template to repair itself via HDR and incorporates the desired corrected bps

Question 15

Why is a silent mutation need to be created at the PAM site when designing HDR and donor templates?

Answer 15

If we don’t HDR could occur but the PAM site would still be intact.
- HDR template cannot contain PAM sites. Another Cas9 could come along and cut the strand undoing the initial work

Question 16

HDR templates require >50 bp homology arms on either side of the point of mutation. True or false?

Answer 16

False, they require >60bp and the longer the better

Question 17

How does a silent mutation affect the PAM site?

Answer 17

It changes the PAM from NGG to NGA, which would still go for the same aa in protein but no longer allows Cas9 to bind to the site

Question 18

Give an example of treating a genetic disease using CRISPR

Answer 18

Duchenne muscular dystrophy (DMD) is caused by an inactivating mutation in the dystrophin gene on X chromosome

• suffers lack normal dystrophin which helps to protect muscles, leading to increased muscle damage and eventual necrosis
• Injecting virus coding for CRISPR/Cas9 improved overall muscle strength in mice

Question 19

Give an example of a theoretical method of creating new antibiotics using CRISPR

Answer 19

1. CRISPR designed to target specific bacterial DNA sequence
2. Molecule is packaged into a bacteriophage carrier to enter target cell
3. System finds gene target, cuts DNA strand at specified location and deletes antibiotic resistance plasmid
4. Targeted cuts of chromosomal DNA always leads to cell death

Question 20

Explain how GM mosquitos can be used to combat malaria

Answer 20

• mosquitos engineered with CRISPR to mutate the double sex (dsx) gene that controls differentiation of sexes
• when released into a population, modified insects will breed with normal mosquitos and pass the dsx gene
• this results in an overall decrease in mosquito population due females’ intersex phenotype (sterility)

Question 21

Explain issues surrounding CRISPR

Answer 21

• other genomic locations may have very high sequence complentarity to the designed gRNA
• manipulation of genomes can be irreversible leading to germline editing (modification being inherited by offspring) and long term effects on population using gene drives