Genome editing

Question 1

What are the 2 double strand break repair mechanisms?

Answer 1

Non-homologous end joining: not perfect, tends to generate indels
Homology directed repair: if we provide the cell with some homologous sequences around the break, the cell will repair the break to look like the provided homologous sequences

Question 2

What is the problem with ends-out and ends-in recombination?

Answer 2

They happen at low efficiency and random integration is still an issue

Question 3

Why can’t we use restriction enzymes for genome editing?

Answer 3

They cut too many times in the genome

Question 4

What are 3 methods of genome editing?

Answer 4

Zinc Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), CRISPR-Cas9

Question 5

What is a zinc finger domain?

Answer 5

Common DNA binding motif in zinc finger transcription factors

Question 6

Do zinc finger domains cut DNA?

Answer 6

No, they act as homing beacons but something else is the scissors

Question 7

What sequences will zinc fingers bind to?

Answer 7

Each one makes contact with 3 specific nucleotides in the major groove in DNA, and we can customize them to bind to whatever sequence we want. We can also link ZFs together to recognize a longer, more specific sequence

Question 8

If ZFs are only the homing beacons, what is the scissors?

Answer 8

Fok1 nuclease domain. Comes from a restriction enzyme but is modular and can act on its own

Question 9

How do we do ZFN genome editing?

Answer 9

Design as many zinc fingers as we need for specific targeting, then link them together. Then attach the Fok1 nuclease domain to the ZFs, and it will cut where the ZFs direct it to

Question 10

What is a TALE domain?

Answer 10

Very specific domain from a bacterial plant pathogen that binds to a single nucleotide

Question 11

What dictates which nucleotide a TALE binds to?

Answer 11

2 particular amino acid residues: the repeat variable di-residues (RVDs)

Question 12

Which 2 amino acids causes a TALE to bind to a C?

Answer 12

HD

Question 13

Which 2 amino acids causes a TALE to bind to a A?

Answer 13

NI

Question 14

Which 2 amino acids causes a TALE to bind to a T?

Answer 14

NG

Question 15

Which 2 amino acids causes a TALE to bind to a G?

Answer 15

NN

Question 16

How do we use TALENs for genome editing?

Answer 16

We can link together specific TALEs to bind to a particular nucleotide sequence, then attach Fok1 nuclease to it to have the TALEs target a specific nucleotide sequence and the nuclease to cleave it

Question 17

What is the CRISPR locus? Where is it?

Answer 17

A string of repeating sequences with spacers of foreign DNA in between the repeats. Found near the Cas genes and the tracrRNA

Question 18

What happens with CRISPR when a bacterial cell survives a viral infection?

Answer 18

Cas proteins will fragment phage DNA near protospacer-adjacent motifs (PAM) into 30 bp fragments, then they get integrated into the 5’ end of the CRISPR locus as spacers once the PAM sequence is cleaved off

Question 19

What happens when the same phage from before tries to infect a bacterial cell again, when the bacteria has a complementary spacer?

Answer 19

The entire CRISPR locus gets transcribed into pre-CRISPR RNA, and the tracrRNA anneals to the repeats. Endogenous RNase III cleaves the pre-CRISPR RNA/tracrRNA into small units with one spacer, one crRNA, and a piece of tracrRNA. Mature crRNA attach to Cas9, and the tracrRNA and the spacer homes in on the complementary piece of invading phage DNA and Cas9 cleaves it