L6 Gene Editing I

Question 1

What is one primary reason scientists use gene editing tools?

Answer 1

to analyse gene function in cells or organisms

Question 2

how can gene editing help study gene regulation?

Answer 2

By turning gene products on or off

Question 3

What application of gene editing involves creating new cellular functions?

Answer 3

Synthetic biology

Question 4

How can gene editing address inherited conditions?

Answer 4

by reversing genetic diseases

Question 5

What type of DNA break is essential for gene editing?

Answer 5

double-stranded DNA (dsDNA) break

Question 6

why are dsDNA breaks significant in cells?

Answer 6

they are toxic and must be repaired to prevent cell death or cancer

Question 7

How do gene editors use DNA repair?

Answer 7

they hijack repair pathways to modify the genome

Question 8

what are the two main dsDNA repair pathways?

Answer 8

non-homologous end joining and homologous recombination

Question 9

Which repair pathway is error-prone and efficient?

Answer 9

NHEJ

Question 10

What type of mutations does NHEJ commonly produce?

Answer 10

INDELs (insertions and deletions)

Question 11

What effect can an INDEL have if it disrupts a reading frame?

Answer 11

it can cause a frameshift and introduce stop codons

Question 12

Where should INDELs be introduced for maximal disruption?

Answer 12

at the start of the gene

Question 13

What happens if an INDEL is a multiple of 3 bases?

Answer 13

the reading frame is preserved

Question 14

Why must edited cells be selected post-NHEJ?

Answer 14

to remove edits that retain the reading frame

Question 15

what phase(s) of the cell cycle is NHEJ active in human cells?

Answer 15

all phases

Question 16

Do all organisms have NHEJ repair?

Answer 16

no, E.coli lacks NHEJ

Question 17

What repair pathway uses a homologous sequence to repair DNA?

Answer 17

homologous recombination

Question 18

how does HR restore the original DNA sequence?

Answer 18

by copying from the undamaged homologous region

Question 19

When is HR active in human cells?

Answer 19

During the S phase of the cell cycle

Question 20

Does E.coli have the HR repair pathway?

Answer 20

yes

Question 21

What do scientists introduce to guide HR-based gene editing?

Answer 21

A repair template with homology flanking a new sequence.

Question 22

Can HR introduce non-natural sequences into cells?

Answer 22

yes

Question 23

What is a limitation of HR editing?

Answer 23

it has lower efficiency than NHEJ

Question 24

What enzyme type is needed to initiate a dsDNA break for editing?

Answer 24

a sequence-specific endonuclease

Question 25

What does KD measure in protein-DNA interactions?

Answer 25

The dissociation constant or binding affinity

Question 26

What must a gene-editing enzyme be specific for?

Answer 26

a unique DNA sequence

Question 27

what is a meganuclease?

Answer 27

an enzyme that recognises long DNA sequences

Question 28

What is an example of a meganuclease?

Answer 28

I-Scel

Question 29

How long is the I-Scel recognition site?

Answer 29

18 base pairs

Question 30

Why is an 18bp recognition site useful?

Answer 30

it appears very frequently in the genome

Question 31

what type of break does I-Scel produce?

Answer 31

a double stranded break

Question 32

Is the I-Scel recognition site naturally present in humans?

Answer 32

no

Question 33

How did scientists test I-Scel in mammals?

Answer 33

by inserting its target site into mouse DNA

Question 34

What did researchers introduce along with I-Scel?

Answer 34

a homologous repair template

Question 35

What competing repair process was observed with I-Scel?

Answer 35

NHEJ

Question 36

Why are meganucleases hard to reprogram?

Answer 36

Small amino acid changes disrupt protein structure

Question 37

What was the next breakthrough after meganucleases?

Answer 37

Zinc finger nucleases (ZFNs)

Question 38

What nuclease domain do ZFNs use?

Answer 38

FokI

Question 39

What does FokI recognise?

Answer 39

The DNA sequence GGATG

Question 40

What kind of DNA cut does FokI produce?

Answer 40

A precise 5' overhang cut downstream of the site

Question 41

What is the role of the zinc finger in ZFNs?

Answer 41

it guides the nuclease to the correct DNA sequence

Question 42

What is the structure of a ZFN protein?

Answer 42

a fusion of zinc finger domains and FokI nuclease

Question 43

How many base pairs does each zinc finger recognise?

Answer 43

3 base pairs

Question 44

How are ZFNs customised for specificity?

Answer 44

by linking multiple zinc fingers for longer recognition.

Question 45

How many zinc fingers are typically used in a ZFN array?

Answer 45

Three or more

Question 46

How many ZFNs are required for a dsDNA break?

Answer 46

two

Question 47

What happens if only one ZFN binds?

Answer 47

A nick is made, which is repaired by ligase

Question 48

How long is the recognition site when two ZFNs bind?

Answer 48

about 18 base pairs

Question 49

Why are ZFNs considered programmable?

Answer 49

They can be designed to target specific sequences

Question 50

What is a major drawback of ZFNs in research labs?

Answer 50

difficult reprogramming and complex design

Question 51

What does FokI require to cut DNA?

Answer 51

Dimerization with another FokI domain

Question 52

What links the zinc finger to the FokI nuclease?

Answer 52

a flexible linker domain

Question 53

What is the role of the linker in ZFNs?

Answer 53

to connect the DNA-binding and cutting domains

Question 54

What are INDELs useful for in gene editing?

Answer 54

Disrupting gene function

Question 55

How are INDELs analysed after editing?

Answer 55

by sequencing the edited region

Question 56

What does a -4 +1 INDEL notation mean?

Answer 56

4 bases were deleted and 1 was inserted

Question 57

Why is gene editing more efficient using NHEJ than HR?

Answer 57

NHEJ is faster and active in all cell cycle phases