CRISPR Flashcards
What is genome-editing?
Cutting genomes using ‘DNA scissors’; CRISPR/Cas9; can be used to remove/add DNA to change the function.
What are genomics?
The study of the entirety of an organism’s genes.
What is Genome-wide association study (GWAS)?
A tool used in genetics research to associate specific genetic variations with particular diseases.
What are functional genomics?
A study that describes gene functions and interactions.
What are ZFNs?
Directed evolution.
What are TALENs?
Clone new RVDs.
When did CRISPR-Cas9 explode into popularity?
2012 onset.
Who discovered the CRISPR sequence?
Yoshizumi Ishino
What does CRISPR stand for?
Clustered Regularly Interspaced Palindromic Repeats
When did the CRISPR win a Nobel prize for genome editing?
2020
What is Cas9?
Protein part of the prokaryotic immune system that defends against viruses,.
What recruits Cas9 to DNA target sites?
tracrRNA:crRNA duplex.
What are the key elements of the CRISPR/Cas9 components?
- Cas9 DNA endonuclease.
- crRNA, which contains 20bp sequence complementary to target.
- Trans-activating crRNA (tracrRNA) which acts as a bridge between crRNA and Cas9 enzyme.
What is PAM?
Protospacer Adjacent motif (NGG)
What is the mechanism of CRISPR/Cas9 DNA cleavage?
- Dna endonuclease targets a DNA sequence via (sgRNA) single guide RNA sequence upstream of the PAM. This results in a 3bp ds break upstream of the NGG.
- Resulting dsbs are repaired by either NHEJ or homology-directed repair (HDR).
What are the off-target effects of CRISPR?
- DNA bulge caused by 1-bp insertion.
2. RNA bulge caused by 1-bp deletion.
What is the target sequence?
The 20 bp upstream the PAM.
What is crRNA?
Crisper RNA; 17-20 nucleotide sequence to target DNA.
Where does Cas9 cleave in the target DNA?
3 bases upstream the PAM.
What is absolutely required for Cas9 cleavage?
The presence of a PAM.
How may CRISPR be delivered? (form)
- DNA/RNA molecule encoding the Cas9 gene.
2. Functional ribunucleoprotein.
How may CRISPR cross the membrane?
- Microinjection
- Electroporatin
- Chemical methods.
What is the function of Cas nickase?
Makes edits in CRISPR to replace target DNA segments (specifed target base) SINGLE STRANDED CUT
What is the function of gRNA?
Targets Cas to a specific locus.
