Interference 2 Flashcards
What is CRISPRi?
A way of tuning down genes.
What is CRISPRa?
An activator uses Dcas9 to be switched on.
How does CRISPRi dCas9 block gene expression?
CRISPR interference uses dead Cas9 that lacks endonuclease activity so binds to DNA but doesnt cut. sgRNA directs dCas9 to the sequence. Acts as a roadblock to transcription. The addition of KRAB enhances gene silencing by actively repressing transcription. Puts human in control of the gene expression.
How has CRISPRi been used?
Exploits the principle of synthetic lethality.
What is synthetic lethality?
When two genes are inactivated together which kills the cell. Must have two mutated to kill the cell.
How do you use synthetic lethality to treat cancer?
Cancer with known mutation screen it with every other mutation in the cell to attempt this.
How can CRISPRi be used in treating teburculosis?
Find two genes to kill the bug.
How does cas12 work?
Cas12 has a single RuvC active site. Performs a staggered cut which makes ssDNA overhangs. Advantageous in CRISPR editing that needs HDR.
How does Cas13 work?
Cuts ssRNA. Has active sites called HEPN higher eukaryote and prokaryote nucleotide binding.
What is a common use of Cas12 and Cas13?
Diagnostics. The kits are called detectr and sherlock.
Why would Cas9 not work for these diagnostics?
Their cutting is too specific.
How does SHERLOCK work?
Specific high-sensitivity enzymatic reporter unlocking. Cas13a is provided with a gRNA matching to the target sequence to be detected. dsDNA or RNA is converted to ssRNA by recombinase polymerase amplification. Cas13-gRNA binds to the target RNA and when it does so the Cas13 promiscuous RNase activity is activated. This activation cuts additional reported RNA molecules in the mixture. Cutting of those releases a FRET signal detectable.
How does DETECTR work?
DNA endonuclease targeted CRISPR Trans reporter. Utilizes Cas12a which cleaves double stranded DNA, which activates its degradation of single stranded DNA. Deployed in lateral flows.
How does RPA work?
How does Casgevy work?
In sickle cell the adult hemoglobin has mutations causing disease. Aim is to repress BCL11A as this blocks the production of fetal hemoglobin in adults. This provides adults with fetal hemoglobin to cure the disease.
