- Duchenne muscular dystrophy (DMD) is caused by a defective dystrophin gene and can be cured by removing a specific faulty exon. If a CRISPR/Cas9 based genome editing approach is to be designed to remove the faulty exon, some of the following conditions have to be met.
A. Two guide RNA molecules to match the flanking regions of the faulty exon
B. An efficient non-homologous end joining system in the host
C. Two separate, specific Cas9 endonucleases to target both ends of the exon
D. A single guide RNA that targets the intron preceding the exoChoose the option that includes all the correct conditions.
(1) A, B and C only (2) A and B only
(3) C and D only (4) A and C onlyThe correct choice is (2) A and B only.
Concept: CRISPR deletion of an exon
To remove a specific exon:
-
Design two gRNAs that bind sequences in the introns flanking that exon.
-
Cas9 makes double-strand breaks (DSBs) at both sites.
-
The cell’s non-homologous end joining (NHEJ) machinery rejoins the cut ends, deleting the intervening exon segment.
Evaluating each condition
A. “Two guide RNA molecules to match the flanking regions of the faulty exon.” – Required
-
One gRNA targets the 5′ intron and the other the 3′ intron around the exon.
-
This is exactly how a clean exon deletion is engineered, so A is correct.
B. “An efficient non-homologous end joining system in the host.” – Required
-
After DSBs, NHEJ is the main repair pathway that ligates the two ends together, excising the exon.
-
Without NHEJ activity, the two breaks would not be joined into a precise deletion; therefore B is correct.
C. “Two separate, specific Cas9 endonucleases to target both ends of the exon.” – Not required
-
A single Cas9 protein guided by two different gRNAs can cut at both flanking sites.
-
There is no need for “two separate Cas9 endonucleases”; standard practice uses one Cas9 and two gRNAs, so C is incorrect.
D. “A single guide RNA that targets the intron preceding the exon.” – Insufficient
-
One cut upstream of the exon would not delete the entire exon; it would only create indels at that one site.
-
To remove the exon, two cuts (5′ and 3′) are needed. Thus, D is incorrect.
Why option (2) is correct
-
The conditions truly necessary for CRISPR/Cas9-based exon deletion are:
-
A: two gRNAs flanking the exon.
-
B: functional NHEJ to join the two cut ends.
-
-
Conditions C and D misdescribe the requirements.
Therefore, the correct set of conditions is A and B only (option 2).
-
