Genetic manipulation 5

Question 1

List 3 types of genome editing

Answer 1

zinc finger nucleases
TALENS
cas9/CRISPR

Question 2

what is the general action of all genome editing techniques?

Answer 2

direct DNA cleaving enzymes to specific sites in any genome where you want to make mutations

Question 3

what is the zinc finger?

Answer 3

protein motif that binds 3bp of DNA

Question 4

normal function of zinc finger

Answer 4

bind specific DNA sequences and activate transcription

Question 5

what DNA nuclease is used?

Answer 5

fok1

Question 6

briefly explain ZFN action

Answer 6

2 ZFN bring 2 fok1 cleavage domains together in gene target
double stranded break - repaired, error prone
random bases inserted, small deletions/insertions

Question 7

what is the error prone repair known as?

Answer 7

non homologous end joining

Question 8

Why are ZFN convenient?

Answer 8

commercial companies design them against target gene
work in vitro and in vivo
good specificity
work on any animal

Question 9

What do TALENs comprise of?

Answer 9

non specific fok1 nuclease domain fused to a customisable DNA binding domain

Question 10

what does TALENs stand for?

Answer 10

transcription activator like effector nucleases

Question 11

what are TALEs?

Answer 11

highly conserved 33-35 bp repeat domains

Question 12

what are TALEs encoded by?

Answer 12

xanthomonas spp proteobacteria

Question 13

what happens to TALEs in the wild?

Answer 13

injected into host plant cells by the bacteria and bind to genomic DNA to alter transcription in host cells

Question 14

What determines the identity of the single base of DNA TALE binds to?

Answer 14

2 hypervariable residues typically found at positions 12 and 13 of the repeat

Question 15

what is cas9?

Answer 15

endonuclease from bacteria - acquired immunity against viruses

Question 16

what bacteria is cas9 from?

Answer 16

streptococcus

Question 17

CRISPR?

Answer 17

clustered regularly interspaced short palindromic repears

Question 18

how is cas9 targeted to cleave DNA?

Answer 18

short RNAs - crRNA and tracr RNA

Question 19

cas/CRISPS system can cut any piece of genomic DNA adjacent to what?

Answer 19

PAM (protospacer adjacent motif)

Question 20

what are PAM’s?

Answer 20

2-5bp sequences that differ between bacterial species

Question 21

what is the PAM for cas9?

Answer 21

GGG

Question 22

the first CRISPR crRNA has a region complementary to what?

Answer 22

part of the target gene, upstream to PAM

Question 23

what does the tracr RNA have a region complementary to?

Answer 23

rest of the crRNA

Question 24

disadvantage of cas9/CRISPR

Answer 24

mutations are uncontrolled and variable

Question 25

how to induce controlled specific mutations in gene of interest using cas9/CRISPR?

Answer 25

catalyse some homology directed repair - electroporate/inject with cas9, sgRNA and a single stranded DNA oligonucleotide homologous to area around breakpoint