lecture 12 - genome editing using CRISP/Cas(

Question 1

genome editing

Answer 1

type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases
- enables specific targeting of sequencing within the genome without impacting the rest of the genome sequence
- potential to cure genetic diseases in patient specific manner

Question 2

what is CRISPR/Cas9

Answer 2

clustered regulatory interspaced short palindromic repeats and CRISPR associated proteins
adaptive immune system of prokaryotes

Question 3

three component system operated as a complex

Answer 3

Cas9
crRNA - crispr rns
tracrRNA
the complex cleaves invading DNA to prevent re infection by viruses

Question 4

CRISPR acts as an immune system in prokaryotes against invading DNA/RNA

Answer 4

1. invading DNA recognised and cut by Cas1-2 protein complexes into fragments called protospacers
2. protospacers intergrated into CRISPR locus located into the bacterial genome

Question 5

CRISPR acts as an immune system in prokaryotes against invading DNA/RNA

Answer 5

1. invading DNA recognised and cut by Cas1-2 protein complexes into fragments called protospacers
2. protospacers integrated into CRISPR locus located into the bacterial genome

Question 6

the protospacers are transcribed to RNA and bind to cas9 protein
when invading strand re infects the complementary Cas9/RNA is recruited and Cas9

Answer 6

cuts DNA strands preventing infection

Question 7

structure of CRISPR locus

Answer 7

transactivating RNA - sequence of RNA needed to bind to the guide RNA to allow cas 9 to bind to its target
then cas operon - encodes for cas protein components
indentical repeat array
spacer of invading DNA

Question 8

the complex formed between the transactivating RNA and the protospacer/CRISPR RNA is called the guide RNA which

Answer 8

enables selective binding of Cas9 to invading DNA sequences

Question 9

Cas9 is a single protein complex

Answer 9

Cas 1 and 2 has many proteins that forms the complex

Question 10

tracrRNA and crRNA form

Answer 10

gRNA

Question 11

protospacer adjacent motifs (PAM) in the invading DNA are required for cas9 to cleave DNA

Answer 11

PAM sequences are not present in the CRISPR locus provides a mechanism to distinguish bacterial self DNA from non-self DNA

Question 12

expression of the protospacer sequences which are called gRNA when they come into contact with the transactivating RNA

Question 13

Genome editing in the lab using CRISPR

Answer 13

we exploit the ability of CRISPR to specifically target and cleave DNA sequences to allow us to modify the genome of cells

Question 14

linker loop

Answer 14

connects the crRNA and tracrRNA together

Question 15

putting the cas9/gRNA complex at a desired locus of the genome will enable site specific cleavage through

Answer 15

nuclease activity

Question 16

the gRNA should contain a protospacer sequence (target sequence) upstream of the

Answer 16

PAM site

Question 17

once the DNA is cleaved the DNA repairs itself by

Answer 17

homology-directed repair
non-homologous end joining

Question 18

homology directed repair only happens in

Answer 18

s phase
this is precise and there will be a sister chromatid to provide repair

Question 19

non homologous end joining

Answer 19

happens everywhere else (primarily in interphase)
DNA repair by NHEJ is error prone
- introduces insertions or deletions into DNA
- impacts gene function
before its ligated
most changes result in premature stop codons can be used for a knock out mutation and normal gene product is not expressed

Question 20

knock in by HDR (introduce a donor template that introduces a new change that we want)

Answer 20

instead of using the sister chromatid for the repair of the DNA we use the donor template we have inserted
remove PAM site so it cant be targeted by future cas9 proteins

Question 21

androgen receptor signalling is a key driver of prostate cancer

Answer 21

current treatment aims to inactivate AR by blocking ligand binding
1. generate prostate cancer cell line expressing cas9 under the control of doxycycline
2. AR gene locus was targeted by CRISPS to create AR knock out cell line

Question 22

could do knock in out stop codon in AR gene