CRISPR

Question 1

what is CRISPR-Cas 9

Answer 1

technology that allows the editing of genomes and gene modification

Question 2

what is CRISPR

Answer 2

Clustered Regularly Interspaced Short Palindromic Repeats

Question 3

what is the CRISPR locus formed of

Answer 3

cas genes
A Ladder sequence
A repeat-spacer array

Question 4

what is the CRISPR locus arrangement

Answer 4

arrangement is not conserved, a genome can have more than one CRISPR, but only one is associated to the cas genes

Question 5

where is CRISPR originated from

Answer 5

viruses attacking prokaryote

Question 6

what do CRISPR form

Answer 6

form part of the antiviral prokaryote defence system or immune system

Question 7

what is CRISPR

Answer 7

sequences in the genomes of prokaryotic organisms

Question 8

what is Cas 9

Answer 8

Crisper associated protein 9

Question 9

what is Cas 9 function

Answer 9

enzyme that uses CRISPR sequences to recognise and cut complementary DNA to these CRISPR sequences

Question 10

what happens in phage infection into cell

Answer 10

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

Question 11

what happens in immunity against reinfection

Answer 11

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

Question 12

where was cas9 first identified

Answer 12

from S. pyrogenes

Question 13

what is cas 9 and what does it contain

Answer 13

endonuclease (cut in middle) that contains

two small RNA molecules

Question 14

what are the two small RNA molecules in cas9

Answer 14

CRISPR RNA (crRNA)
transactivating CRSPR RNA

Question 15

what was formed from the manipulation of the two single strands, what did this allow

Answer 15

“single-guide RNA” (sgRNA)

Cas9 was then able to find and cut a target sequence

Question 16

what is PAM

Answer 16

Protospacer Adjacent Motifs

Question 17

what is PAM function

Answer 17

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

Question 18

what is the conservation of PAM like

Answer 18

conservation of PAM varies in the different CRSPR-Cas systems and they may evolved with the Cas1 and the leader sequence

Question 19

what are the uses of CRISPR

Answer 19

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

Question 20

what are examples of modification of organisms using CRISPR-cas9

Answer 20

Yeast has been modified to make biofuels
Plants have been modified to improve crops.
Animals modified e.g. create Mosquitos “anti-malaria”

Question 21

what is crucial in the use of CRISPR

Answer 21

in any other genetic study the inclusion of controls, both positive and negative are crucial

Question 22

what happens in DN-free CRISPR-cas9 gene editing

Answer 22

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