Genet 302 lec 53
What kind of DMD deletions usually cause Becker muscular dystrophy?
In-frame deletions
What kind of DMD deletions usually cause Duchenne muscular dystrophy?
Out-of-frame deletions
What mutation was emphasized in this lecture?
Deletion of exon 44
Why does deletion of exon 44 cause Duchenne muscular dystrophy?
It disrupts the reading frame
What strategy that worked in mice was reviewed at the start of the lecture?
A DNA base editor was used to cause exon 45 skipping so exon 43 connected to exon 46
What patient cells were used to begin the human repair workflow?
Peripheral blood mononuclear cells
What were those patient blood cells turned into for the experiment?
iPS cells
What was the CRISPR target in the DMD correction strategy?
The splice site around exon 45
What CRISPR editing tool was used in this lecture?
An adenine base editor
How does ABE-CRISPR make a specific base-pair substitution?
It targets a chosen adenine and converts A·T into G·C without making a double-strand break
What base change was used to destroy the splice site in this lecture?
A to G
How is the A to G change completed after ABE acts?
Through DNA repair or DNA replication
What was the purpose of destroying the exon 45 splice site?
To make exon 45 be skipped during splicing
How can exon skipping turn an amorphic allele into a hypomorphic allele?
Skipping an exon can restore the reading frame, allowing production of a shorter but partly functional protein
In the ΔEx44 example, what new exon connection is made after exon 45 skipping?
Exon 43 connects to exon 46
Why is that helpful in ΔEx44?
It restores the reading frame
Did corrected iPS cells themselves make dystrophin protein?
No
Why did corrected iPS cells not make dystrophin yet?
Because dystrophin is turned on in muscle cells, not in iPS cells
What were the corrected iPS cells turned into next?
Heart muscle cells
What happened after the corrected cells were turned into heart muscle cells?
Dystrophin expression was restored
What is the main testable idea about ABE-CRISPR from this lecture?
It can make specific base substitutions to alter a splice site
What is the main testable idea about exon skipping from this lecture?
It can convert a severe amorphic allele into a milder hypomorphic allele by restoring the reading frame