CRISPR and genetically engineering mice

Question 1

CRISPR is used to

Answer 1

knock-in/ knock out genes

Question 2

Knock-outs

Answer 2

helps establish function of gene

Question 3

knock-ins

Answer 3

introduces specific mutations

Question 4

what does CRISPR use

Answer 4

Cas9 technology

Question 5

CRISPR can be used to

Answer 5

• cure diseases e.g. CF

- allows is to genetically modify organism

Question 6

simple process of how CRISPR works

Answer 6

works by targeting determined sequences (genes) and then the CRISPR machinery recruits Cas9 which cleaves DNA at target site

Question 7

Cas9 is a

Answer 7

nuclease

Question 8

CRISPR process

Answer 8

1) sgRNA is introduced which has an identical sequence to the target gene
2) sgRNA also contains structures which recruit Cas9 once it is bound to the target sequence
3) sgRNA base pairs with target sequence
4) Cas9 ides to tarte sequence and cleaves through both strands of DNA
5) leaving a straight break

Question 9

sgRNA

Answer 9

small guide RNA

Question 10

what is designed so CRISR can be used in cells

Answer 10

a plasmid is designed containing Cas9 binding site downstream of a promotor

Question 11

then plasmid is introduced to a target cell via

Answer 11

transduction or transfection

Question 12

how to breed genetically modified mice

Answer 12

1) order or design and produce ES cells
2) microinjection into fertilised oocyte into blastocysts
3) transfer to pseudopregnant females
4) birth of litter
5) breed chimeric odd spring with wild-type mice
6) screen for gremlin transmission
7) expansion

Question 13

what stage of oocyte is the ES injected into

Answer 13

Blastocyst

Question 14

GM oocyst is transferred to

Answer 14

a suedopregannt female

Question 15

chimeric offspring are then bred with

Answer 15

wild-type mice

Question 16

Transfection

Answer 16

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.