CRISPR Flashcards
what is CRISPR-Cas 9
technology that allows the editing of genomes and gene modification
what is CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
what is the CRISPR locus formed of
cas genes
A Ladder sequence
A repeat-spacer array
what is the CRISPR locus arrangement
arrangement is not conserved, a genome can have more than one CRISPR, but only one is associated to the cas genes
where is CRISPR originated from
viruses attacking prokaryote
what do CRISPR form
form part of the antiviral prokaryote defence system or immune system
what is CRISPR
sequences in the genomes of prokaryotic organisms
what is Cas 9
Crisper associated protein 9
what is Cas 9 function
enzyme that uses CRISPR sequences to recognise and cut complementary DNA to these CRISPR sequences
what happens in phage infection into cell
phage DNA inserted into cell cas1 and cas2 cut the DNA protospacer acquisition spacer integration the DNA that was cut is added to the bacteria chromosome
what happens in immunity against reinfection
transcription occurs
DNA segment binds to cas 9
compare the sequence of DNA that is inserted by virus and is cut - cleavage if the same
where was cas9 first identified
from S. pyrogenes
what is cas 9 and what does it contain
endonuclease (cut in middle) that contains
two small RNA molecules
what are the two small RNA molecules in cas9
CRISPR RNA (crRNA) transactivating CRSPR RNA
what was formed from the manipulation of the two single strands, what did this allow
“single-guide RNA” (sgRNA)
Cas9 was then able to find and cut a target sequence
what is PAM
Protospacer Adjacent Motifs
what is PAM function
spacers from viral nucleic acids that prokaryotes select to integrate in their CRISPR locus, they are found adjacent to short DNA sequences of 3-5 bp
what is the conservation of PAM like
conservation of PAM varies in the different CRSPR-Cas systems and they may evolved with the Cas1 and the leader sequence
what are the uses of CRISPR
Gene silencing
Gene editing
Transient gene silencing or transcriptional repression (CRISPRi)
Transient activation of endogenous genes (CRISPRa or CRISPRon)
Embryonic stem cell and transgenic animals have been generated with this system
Pooled genome-scale knockout screening
Modification of organisms
Re-evaluations related to gen-disease claims have been carried out
what are examples of modification of organisms using CRISPR-cas9
Yeast has been modified to make biofuels
Plants have been modified to improve crops.
Animals modified e.g. create Mosquitos “anti-malaria”
what is crucial in the use of CRISPR
in any other genetic study the inclusion of controls, both positive and negative are crucial
what happens in DN-free CRISPR-cas9 gene editing
day 1
- cas9 protein or cas9 mRNA, crRNA-tracrRNA and co-transfection added to plate
day 3
- analyse gene editing
- clonal cell line isolation and expansion
3-6weeks
- analyse phenotype
