12.2 CRISPR

Question 1

Spacer acquisition (adaption)

Answer 1

Recognise spacers in the bacteriophage genome and excise them. With the help of PAM.

Question 2

Expression of cRNAs

Answer 2

Spacers and repeats are expressed to make precursor crRNAs to guide the immune response.

Question 3

Interference

Answer 3

Mature crRNAs with cas9 look for complementarity in the invader genome to then cleave and degrade it.

Question 4

Protospacer adjacent motif

Answer 4

Any NNG next to the spacer sequence - common but not found within the CRISPR DNA array

Question 5

Key innovation of CRISPR

Answer 5

Substitution of chimeric gRNA in place of natural crRNA and tracrRNA. These sequences are specific to a target sequence in the genome to be edited.

Question 6

Genome editing with CRISPR-Cas

Answer 6

sgRNA assembles with Cas9 (effector complex) that then finds PAM and unwinds upstream DNA to pair with it and make a double stranded cut.

Question 7

Cellular DNA repair mechanisms

Answer 7

Without template - no homologous end joining.
Using template - homology directed repair.

Question 8

Non-homologous end-joining

Answer 8

Nucleotides randomly inserted or deleted as the cleaved ends are rejoined (commonly creates INDELs). If no INDEL occurs then Cas9 keeps cutting until it does to create a frameshift mutation - gene silencing “knock-out”

Question 9

Homology directed repair

Answer 9

Same repair enzymes as crossing over, uses homologous chromosomes as a template.

Question 10

CRISPR-cas9 advantages

Answer 10

Cheap
Targeted
Specific
Creates knock-outs
Used on live cells
Stimulate with donor DNA - HDR.

Question 11

Challenges of CRISPR

Answer 11

Off-target effects
Hard to control NHEJ or HDR
Mosaicism

Question 12

Potential uses of CRISPR

Answer 12

Disrupt genes to determine function
Editing - reverse mutations donor organs, improve animals, domestication, de-extinction, gene drives.