L2 - Genome editing using CRISPR/Cas9

Question 1

What does the human genome contain?

Answer 1

Contains approx. 3 billion bp, which reside in the 23 pairs of chromosomes within the nucleus of all our cells (exceptions being RBCs & gametes)

Question 2

What is genome editing?

Answer 2

A type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases (‘molecular scissors’)

Potential to cure numerous currently untreatable genetic diseases and also certain cancers without the use of traditional drug compounds

Question 3

When was PCR invented?

Answer 3

1985

Question 4

When was CRISPR-mediated genome editing invented?

Answer 4

2012

Question 5

What was the first genome sequenced?

Answer 5

Influenza virus - 1995

Question 6

What does CRISPR/Cas system stand for?

Answer 6

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) & CRISPR associated (Cas) proteins

Question 7

History of the CRISPR/Cas system

Answer 7

CRISPR described as an anti-viral ‘adaptive immunity’ defence mechanism in prokaryotes (2007)

Cas9 first used for targeted genome editing in prokaryotes by Prof. Emmanuelle Charpentier & Prof. Jennifer Doudna (2012)

Cas9 protein adapted for use in eukaryotes by Prof. Feng Zhang (2013)

Question 8

CRISPR in prokaryotes

Answer 8

The CRISPR/Cas system is a prokaryotic ‘immune system’ – defence against invading plasmids & phages

CRISPR associated proteins (Cas) use the CRISPR spacers to recognise and cut the exogenous genetic elements

CRISPRs are found in approx. 40% of sequenced bacterial genomes & 90% of sequenced archaea

Question 9

What is PAM?

Answer 9

PAM – Protospacer Adjacent Motif

In order for Cas9 to function, it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene

Question 10

What is NHEJ?

Answer 10

Non-Homologous End Joining

DNA repair mechanism

Question 11

Why is NHEJ not very accurate?

Answer 11

NHEJ is error prone & thus can cause mutations that ‘knock out’ genes either by loss of coding DNA or by shifting the translational codon reading frame

Question 12

What is HDR?

Answer 12

Homology Directed Repair

HDR uses any complementary DNA flanking the breakage point as a template to synthesise fresh DNA to repair the cut strands

Less error prone but slower than NHEJ

Essential for preventing cancer

Question 13

CRSIPR as a genome editing tool – KI

Answer 13

HDR template requires >60bp homology arms on either side of the point of mutation/insertion – the longer the better

HDR template cannot contain PAM sites – must create silent mutations

Inserts of several kbps are possible

Question 14

What is Duchenne muscular dystrophy (DMD)?

Answer 14

A genetic disease caused by an inactivating mutation in the dystrophin gene on the X chromosome (locus Xp21)

Humans with the condition lack normal dystrophin, which helps to protect muscles from injury

This leads to increased muscle damage & eventual necrosis of muscle tissue

By injecting the virus coding for CRISPR/Cas9 designed to excise the disease-causing mutation into mice with DMD, overall muscle strength was improved (but not cured)

Question 15

New antibiotics using CRISPR

Answer 15

1. CRISPR is designed to target a specific bacterial DNA sequence
2. The molecule is packaged into a bacteriophage carrier to enter the target cell
3. The system finds the gene target, cuts the DNA strand at the specified location, & deleted the antibiotic resistance plasmid
4. Targeted cuts of chromosomal DNA always lead to cell death

Question 16

What causes malaria?

Answer 16

Caused by the parasite Plasmodium falciparum, which uses mosquitos as a carrier to infect humans

Question 17

GM mosquitos to combat malaria

Answer 17

Mosquitos engineered with CRISPR/Cas9 to mutate the doublesex (dsx) gene that controls differnation of the 2 sexes

When released into a population, modified insects will breed with normal non-modified mosquitos & pass the mutated sex-determined gene onto their offspring

Results in an overall decreases of the population due to females’ intersex phenotype = sterile

In the lab this led to a whole mosquito population being wiped out within 7-11 generations

‘Gene Drive’ showed 100% prevalence before population collapse – always given on to offspring

Question 18

Caveats of CRISPR

Answer 18

Potential of off-target effect mutations - more specific Cas proteins are being developed

Ethical considerations regarding the irreversible manipulation of genomes

Delivery of CRISPR to target very specific tissues, but not others, in animals is not 100% efficient yet

Home ‘gene editing’ kits becoming available

Ongoing patenting lawsuits

Question 19

Where does the CRISPR system originate from?

Answer 19

A prokaryotic viral defence mechanism (similar to RNAi in eukaryotes)

Question 20

How do Cas nucleases know where to target in the genome?

Answer 20

Cas nucleases require guide RNAs (gRNAs) & protospacer adjacent motifs (PAMs) to target them to a specific location within the genome

Question 21

What happens when Cas cuts the DNA?

Answer 21

Cas-mediated cutting of DNA triggers the cells intrinsic DNA repair mechanism

Question 22

What are the 2 ways DNA repair can occur?

Answer 22

Non-homologous end joining (NHEJ)

Homology-directed repair (HDR)

Question 23

What are the outcomes possible after NHEJ has occurred?

Answer 23

It could all fix back together as before as the wild type (30-40% of the time)

Insertion of bases

Deletion of bases

Frameshift mutation after the wrong amino acids/protons have been transcribed

Question 24

How does CRISPR use NHEJ?

Answer 24

To disrupt or knock out specific genes

Question 25

How does CRISPR use HDR?

Answer 25

CRISPR-induced HDR allows for introduction of specific template DNA – eg. desired point mutations/ protein tagging