10 - Gene Editing

Question 1

What is gene editing?

Answer 1

Precise, targeted modification of an organism’s DNA to create indels, point mutations, or HDR-mediated insertions

Question 2

What does gene editing not require?

Answer 2

Embryonic stem cells, because it edits endogenous genes directly

Question 3

What are indels?

Answer 3

Insertions or deletions generated mainly through NHEJ

Question 4

What are the two main DNA repair pathways involved in gene editing?

Answer 4

NHEJ and HDR

Question 5

Why is NHEJ considered error-prone?

Answer 5

It repairs DSBs quickly and randomly, often producing indels

Question 6

Why is HDR limited?

Answer 6

It’s efficient only in dividing cells and requires a donor template

Question 7

What is the general mechanism of gene editing?

Answer 7

Deliver nuclease → target nuclease → create DSB → repair via NHEJ or HDR

Question 8

Where does Cas9 cut relative to the PAM?

Answer 8

About three base pairs upstream of the PAM

Question 9

What problem do double-strand breaks cause?

Answer 9

Toxicity and reduced cell survival

Question 10

What phenotype was seen in the yeast ADE2 experiment?

Answer 10

Pink colonies showed successful editing with loss of ADE2 function

Question 11

What gene-editing tools existed before CRISPR?

Answer 11

ZFNs and TALENs

Question 12

Why were ZFNs difficult to use?

Answer 12

Each target required custom-engineered proteins, making them slow, expensive, and complex

Question 13

What made CRISPR/Cas9 revolutionary?

Answer 13

Only the guide RNA needs changing, making it fast, cheap, and highly efficient

Question 14

What is the biological origin of CRISPR?

Answer 14

Bacterial adaptive immunity using stored viral spacers to recognise and cut invading DNA

Question 15

What is a PAM sequence?

Answer 15

A short motif (NGG for Cas9) required for Cas9 recognition and binding

Question 16

What does the sgRNA do?

Answer 16

Guides Cas9 to a complementary DNA sequence for cutting

Question 17

What are the basic steps in a CRISPR experiment?

Answer 17

Design gRNA → deliver CRISPR components → identify edited organisms

Question 18

How can CRISPR components be delivered?

Answer 18

Via plasmid DNA, mRNA, or ribonucleoprotein complexes using transformation, transfection, or microinjection

Question 19

How can you detect CRISPR edits?

Answer 19

Phenotype, RFLP, sequencing, or selection markers

Question 20

What is a Cas9 nickase?

Answer 20

Cas9 mutated to cut only one DNA strand

Question 21

What is dCas9?

Answer 21

Catalytically dead Cas9 that binds DNA without cutting

Question 22

What can dCas9 be used for?

Answer 22

RISPRi, CRISPRa, epigenetic editing, and fluorescent DNA labelling

Question 23

What is the purpose of base editing?

Answer 23

Change one DNA base into another without creating a DSB

Question 24

What enzymes are fused to Cas9 in base editors?

Answer 24

Cytidine deaminase for C→T and adenosine deaminase for A→G editing

Question 25

Why are base editors less toxic?

Answer 25

They avoid double-strand breaks and minimise indels

Question 26

What is prime editing used for?

Answer 26

Introducing point mutations, small insertions, or deletions without DSBs or donor templates

Question 27

What does the pegRNA contain?

Answer 27

Targeting sequence, primer binding site, and reverse-transcription template

Question 28

How does prime editing work?

Answer 28

nCas9 makes a single-strand nick → RT writes new sequence into DNA → cell incorporates the edit