Gene editing Flashcards
What are the methods of gene editing?
Zalen, Talen, CRISPR
What is genome editing?
It is engineered nuclease that inserts, deletes, replaces specific targeted sequences.
What is the engineered nuclease made up of?
Non specific cleavage module-induce double stranded breaks
Specific DNA binding domains
What are the products of the double stranded breaks induced by engineered nuclease? What kind of modification is associated with each?
Non homologous recombination- targeted mutations or deletions
Homologous recombination- Targeted replacement/insertion
What is the ZFN nuclease made up of?What is in the transfection?
It has DNA cleaving domain-Fok1
It has a DNA binding domain- zinc finger protein
Fusion protein
What is Talen nuclease made up of? What is in the transfection?
Tale- DNA binding domain
Fok1- DNA cleaving domain
Fusion protein
How does CRISPR modify DNA? What advantage does it have over the other two techniques and what disadvantage does it have?
It has CAS9 enzyme(that is lysine rich for NLS) that is targetted by gRNA cleaves PAM sequences and inserts sequence. Much more control over the modification. Multiple targeting possible. It has higher risk for off target effects but less specific
What is the nuclease for CRISPR? What is the DNA binding protein? What is the vector made up of?
cas9, gRNA, protein and RNA
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
What does the CRISPR locus have? What is it originally from and why is this system there?
TransRNA, CAS 9 protein complex, and spacers and repeats.
Bacteria
It is protection from viral infections
What are spacers?
They are viral DNA that is incorporated into the genome as repeats.
Explain the CRISPR system in bacteria?
A cell gets viral infection, CAS9 gets expressed and cleaves the viral DNA, and then the fragments get incorporated into the genome as spacers between repeats. When a second infection occurs, spacers and repeats are transcribed as pre-cRNA and tracRNA and CAS 9 is also transcribed it forms a complex by hybridizing and then the complex is processed and matures and then the viral DNA is recognized via the spacers, while CAS9 cleaves the viral DNA.
What does CAS 9 cleave? What recognizes the target sequence?
target sequence that is upstream of PAM(NGG or NAG), the GRNA has a 20 NT that recognizes it.
What are some applications of CRISPR-cas9?
Gene editing, visualization(fluorescence), regulation(t.f), reorganization
Why is CRISPR less specific than other genome editing techniques? What are the consequences?
Because it can tolerate1-3 base pair mismatches. They can lead to oncogenesis through double stranded breaks that happen off target causing mutations in these genes
