L2 - Genome editing using CRISPR/Cas9 Flashcards
What does the human genome contain?
Contains approx. 3 billion bp, which reside in the 23 pairs of chromosomes within the nucleus of all our cells (exceptions being RBCs & gametes)
What is genome editing?
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
When was PCR invented?
1985
When was CRISPR-mediated genome editing invented?
2012
What was the first genome sequenced?
Influenza virus - 1995
What does CRISPR/Cas system stand for?
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) & CRISPR associated (Cas) proteins
History of the CRISPR/Cas system
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)
CRISPR in prokaryotes
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
What is PAM?
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
What is NHEJ?
Non-Homologous End Joining
DNA repair mechanism
Why is NHEJ not very accurate?
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
What is HDR?
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
CRSIPR as a genome editing tool – KI
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
What is Duchenne muscular dystrophy (DMD)?
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)
New antibiotics using CRISPR
- CRISPR is designed to target a specific bacterial DNA sequence
- The molecule is packaged into a bacteriophage carrier to enter the target cell
- The system finds the gene target, cuts the DNA strand at the specified location, & deleted the antibiotic resistance plasmid
- Targeted cuts of chromosomal DNA always lead to cell death
What causes malaria?
Caused by the parasite Plasmodium falciparum, which uses mosquitos as a carrier to infect humans
GM mosquitos to combat malaria
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
Caveats of CRISPR
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
Where does the CRISPR system originate from?
A prokaryotic viral defence mechanism (similar to RNAi in eukaryotes)
How do Cas nucleases know where to target in the genome?
Cas nucleases require guide RNAs (gRNAs) & protospacer adjacent motifs (PAMs) to target them to a specific location within the genome
What happens when Cas cuts the DNA?
Cas-mediated cutting of DNA triggers the cells intrinsic DNA repair mechanism
What are the 2 ways DNA repair can occur?
Non-homologous end joining (NHEJ)
Homology-directed repair (HDR)
What are the outcomes possible after NHEJ has occurred?
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
How does CRISPR use NHEJ?
To disrupt or knock out specific genes
How does CRISPR use HDR?
CRISPR-induced HDR allows for introduction of specific template DNA – eg. desired point mutations/ protein tagging
