Lectures 13&16 - Genome editing, CRISPR and functional genomics

Question 1

which 3 components are used in CRISPR? (1 protein and 2 RNA)

Answer 1

Cas9 - protein
crRNA, tracrRNA

Question 2

in prokaryotes, what is the function of CRISPR?

Answer 2

cleave invading DNA from viruses to prevent their re-infection

Question 3

invading viral DNA is cleaved in CRISPR. By which two Cas molecules? into what?

Answer 3

cut by Cas1 and Cas 2 into protospacers (fragments)

Question 4

upon viral reinfection to bacterial cells which have been treated by CRISPR, what happens?

Answer 4

transcription of protospacers to RNA

RNA binds Cas9

RNA/Cas9 complex is recruited to invading DNA (on the complementary strand)

Cas9 cuts DNA strands creating a ds break to stop infection - NHEJ occurs (knock out)

Question 5

the CRISPR locus contains which 3 components?

which molecule, not on the CRISPR locus, helps enable cleavage?

Answer 5

tracrRNA
Cas operon
repeat spacer array

PAMs enable Cas9 cleavage

Question 6

CRISPR has been used to edit which two genes (2 separate diseases)?

Answer 6

AR receptor gene for prostate cancer

CCR5 gene for HIV

Question 7

illumina sequencing reactions measure what?

what produces the component which is measured?

Answer 7

the colour of the light emitted from the reaction

DNA bases are labelled in different colours so produce the colour signals

Question 8

what is the correct order for RNA sequencing approaches?
a - data analysis
b - RNA extraction
c - sequencing
d - library prep

Answer 8

B, D, C, A

Question 9

What does CHIP sequencing determine? what does it require?

Answer 9

where a protein binds to DNA

requires a high quality antibody

Question 10

ATAC sequencing relies on a ……… to reveal accessible regions of …….

Answer 10

transposase

chromatin

Question 11

bisulphite sequencing measures what?

how does it measure this?

what reaction occurs?

Answer 11

the methylation of DNA

detects the base change of unmethylated C to T

via a deamination reaction

Question 12

RRBS sequencing uses which restriction enzyme?

what is its recognition site/ what does it enrich for?

Answer 12

uses MspI enzyme

enriches CpG islands, recognises CCGG sequences

Question 13

which 4 components are required to produce transgenic models?

Answer 13

gene of interest, promoter, 3’ protein tag and poly A tail

Question 14

which recombinase enzyme catalyses the recombination of LoxP sites?

Answer 14

cre recombinase

Question 15

which enzyme catalyses the recombination of FRT sites?

Answer 15

flippase

Question 16

knock in mouse models have been used for which disease?

Answer 16

multiple epiphyseal dysplasia