Lecture 33: CRISPR and Gene Editing Techniques Flashcards
What is Genome editing?
Cutting the genome by DNA scissors, CRISPR/Cas9
What can genome editing be used for?
Removing changing and adding DNA
What is Genomics?
The study of the entirety of an organism’s genes using bioinformatics to find DNA-sequence variations that affect health, disease or drug response
What are Genome-wide association studies (GWAS) used for?
To associate specific genetic variations with particular diseases
What is functional genomics?
A field of molecular biology that attempts to describe a gene (and protein) functions and interactions
What are the differences between the gene editing tools?
They all perform the same function but newer ones are easier to program
What is the advantage to CRISPR-Cas9?
It is easy to design and generate gRNA that binds to target sequence
What were TALENs used to do?
Clone new RVDs
Where was CRISPR/Cas9 found?
In prokaryotes
What does CRISPR/Cas9 do in prokaryotes?
It is part of the prokaryotic immune system that defends against viruses. CRISPR/Cas9 will recognize embedded DNA from viruses and incorporate it in the bacteria’s genome to recognize and inactivate it by cutting it
What is Cas9 recruited to the DNA target site by?
The duplex tracerRNA:crRNA (guide RNA)
What are the three key elements of CRISPR/Cas9?
- the Cas9 DNA endonuclease
- cRNA which contains a 20 bp sequence complementary to the target
- a trans-activating crRNA (tracer RNA) which acts as a bridge between the crRNA and the Cas9 enzyme
What is the cRNA in Crispr?
A 20 BP sequence complementary to the target
What does the trans-activating crRNA (tracrRNA) do?
Acts as a bridge between the crRNA and the Cas9 enzyme
How is DNA endonuclease targeted to a DNA sequence?
Via a single guide RNA (sqRNA) sequence upstream of the PAM
What does DNA endonuclease result in?
A double-stranded break
What are the two types of double-stranded breaks caused by DNA endonuclease?
Non-homologous end joining (NHEJ) or Homology-directed repair (HDR)
What is a PAM sequence?
A three bp sequence for the CRISPR/Cas9 to target (NGG)
What is the issue with NHEJ?
During repair, genes are often disrupted by insertions or deletions so a lot of errors occur
How does HDR work?
It uses gene replacement or correction by homologous recombination through the complementary chromosome
What are the issues of CRISPR/Cas9?
Off-target effects where DNA cleavage occurs elsewhere
Why is PAM important?
The 3’ end of the DNA sequence must have a protospacer adjacent motif (PAM) (5’-NGG-3’) because this is how the CRISPR will target it 20 nucleotides upstream
What is the PAM not included in?
The sgRNA
How can Cas9 be delivered?
As DNA, mRNA or functional ribonucleoprotein (RNP)
What is the biggest challenge when delivering CRISPR?
Delivering the cargo across the cell membrane
How can HDR and CRISPR be used for gene editing?
CRISPR can be used to cut DNA and then a ssDNA Donor Template can be used for HDR to create precise gene knock-in
What is Base Editing?
Instead of creating double-stranded breaks, it can take single base pairs in the genome
How does Base Editing work?
Cas nickase or Cas fused to a deaminase makes the edit by using a gRNA to target a specific locus
Why is Base Editing better?
Because it doesn’t introduce double-stranded breaks so there is less propensity for error
What do Prime-editors do?
Used an engineered reverse transcriptase fused to Cas9 nickase and a prime-editing guide RNA (pegRNA) to change the base pairs
What occurs in CRISPRa?
Cas9 can be fused to other proteins that can activate gene expression
What occurs in CRISPRi?
Cas9 can be fused to proteins that can interfere with gene expression
How does Epigenome editing work?
A dCas9 fused to the core catalytic domain of p300 acetylates target sites in the genome. Acetylation of the target gene synergizes with the action of transcription factors and RNA polymerase II resulting in transcriptional upregulation
What is CRISPR screening/CRISPR Library?
Using CRISPR to identify genes that are resistant to drugs, or toxins, or are tumours to create mutations in them
What is in vivo?
Work being conducted in living organisms
What is ex vivo?
Studies conducted on functional organs
Which diseases are treated in-vivo?
Diseases of the eye and ear
What is Ex-vivo therapy useful for?
Blood diseases sickle cell anemia, hemophilia CAR T cancer
What is Somatic Gene editing?
Editing any cell in a living organism other than a reproductive cell not passed onto offspring
What is Germline Gene editing?
Editing cells segregated and involved in the reproductive process that are passed on to progeny
