CRISPR & Cas-9

Question 1

What are the two essential components required for CRISPR–Cas9 gene editing in the lab?

Answer 1

1.) Cas9 protein
2.) Single-guide RNA (sgRNA) containing a 20-bp targeting sequence + scaffold

Question 2

How does Cas9 locate the correct place in the genome to cut?

Answer 2

Cas9 first scans DNA for a PAM sequence (NGG) → then checks for complementarity between sgRNA and the adjacent DNA. Only if both match does it unwind and cut.

Question 3

Why is the PAM sequence essential?

Answer 3

Cas9 will not bind or cut without PAM. PAM acts as a checkpoint ensuring Cas9 targets foreign DNA, not bacterial host DNA.

Question 4

What are the two repair pathways that follow a Cas9-induced double-strand break?

Answer 4

1.) NHEJ (Non-Homologous End Joining): Error-prone, introduces indels → gene knockout
2.) HDR (Homology-Directed Repair): Requires template → precise edits or insertions

Question 5

Why does CRISPR editing work best in dividing cells?

Answer 5

HDR is active during S/G2 phases, so precise edits depend on cell division. Non-dividing cells rely mostly on NHEJ.

Question 6

What experiment demonstrated that Cas9 needs sgRNA to cut DNA?

Answer 6

In-vitro reconstitution:
Cas9 + sgRNA + DNA → cut
Cas9 alone or Cas9 + DNA without sgRNA → no cut

Question 7

What does dCas9 do?

Answer 7

Dead Cas9 binds DNA but cannot cut. Used for CRISPRi (gene silencing) or CRISPRa (gene activation)

Question 8

What causes off-target effects in CRISPR editing?

Answer 8

sgRNA binding to similar, but not identical, DNA sequences → unintended cuts

Question 9

How does CRISPR differ from RNAi?

Answer 9

CRISPR edits DNA permanently
RNAi silences mRNA transiently

Question 10

What is a missense mutation?

Answer 10

A single nucleotide change resulting in one amino acid substitution

Question 11

What mutation usually creates loss-of-function through frameshifts?

Answer 11

Indels not in multiples of 3

Question 12

What is a nonsense mutation?

Answer 12

A mutation that introduces a premature stop codon

Question 13

What does the sgRNA do?

Answer 13

Guides Cas9 to the target DNA via base-pairing

Question 14

Where is the target sequence relative to the PAM?

Answer 14

The 20 bp guide lies directly upstream of the PAM

Question 15

What is the original purpose of CRISPR in bacteria?

Answer 15

Adaptive immunity against viruses

Question 16

How is CRISPR different from restriction enzymes?

Answer 16

Restriction enzymes recognize fixed DNA sequences; CRISPR is programmable

Question 17

In vivo editing faces what major issue?

Answer 17

Delivery to tissues

Question 17

Which steps are performed by Cas9?

Answer 17

Binding the target and cutting the DNA

Question 18

Which steps rely on the cell?

Answer 18

DNA repair (NHEJ or HDR)

Question 19

Which repair pathway introduces indels?

Answer 19

NHEJ

Question 20

Which repair pathway enables knock-in edits?

Answer 20

HDR

Question 21

What is dCas9 used for?

Answer 21

Binding DNA without cutting; used for regulation or labeling

Question 21

What do base editors do?

Answer 21

Convert one nucleotide to another without causing DSBs

Question 22

How do you make a large deletion with CRISPR?

Answer 22

Use two sgRNAs flanking the region

Question 23

How do you create a knock-in mutation?

Answer 23

Use HDR with a donor template

Question 24

Name a CRISPR therapy for sickle cell disease & how its done

Answer 24

Casgevy 1. Extract hematopoietic (blood) stem cells from patient’s bone marrow. 2. Gene-edit them in the lab. 3. Reimplant edited cells → produce normal red blood cells

Question 25

What is the biggest technical barrier to CRISPR therapy?

Answer 25

Efficient and safe delivery in vivo

Question 26

Why is HDR difficult in adult tissues?

Answer 26

Most cells are non-dividing and HDR is active mainly in S/G2