Genet 302 lec 31 & 32
What are the 2 main types of muscular dystrophy emphasized at the start of lecture 31?
Becker muscular dystrophy and Duchenne muscular dystrophy
What kind of DMD deletion usually causes Becker muscular dystrophy?
An in-frame deletion
What kind of DMD deletion usually causes Duchenne muscular dystrophy?
An out-of-frame deletion
Why is deletion of exon 50 a common Duchenne mutation model?
Because deleting exon 50 shifts the reading frame so exon 51 contains a stop codon
What was the first goal of the 2022 CRISPR paper in lecture 31?
To make a humanized knock-in mouse model of Duchenne muscular dystrophy
How was the humanized DMD mouse model made?
By replacing mouse exon 51 with human DMD exon 51, then deleting mouse exon 50
What was inserted in step 1 of the mouse-model construction?
Human DMD exon 51
What was deleted in step 1 of the mouse-model construction?
Mouse exon 51
What CRISPR strategy was used in step 1 to replace mouse exon 51 with human exon 51?
Knock-in by HDR in zygotes
How many guide RNAs and donor DNAs were used in step 1?
2 guide RNAs and 1 donor DNA
What was done in step 2 of the mouse-model construction?
Mouse exon 50 was deleted
What CRISPR strategy was used in step 2 to delete exon 50?
Knockout-style deletion by CRISPR in zygotes- NHEJ
How many guide RNAs and donor DNAs were used in step 2?
2 guide RNAs and 0 donor DNAs
What successful mouse strain was produced after steps 1 and 2?
A humanized Δ50 mouse, called h51KI/Δ50
Why was cDNA sequencing used to verify the successful humanized strain?
To show the edited mRNA had the expected exon structure and remained in frame
What phenotype was seen in the humanized Δ50 mouse before treatment?
Loss of dystrophin in muscle and heart
What was the second goal of the 2022 paper in lecture 31?
To correct the humanized Duchenne mouse model by CRISPR-Cas9 therapeutic editing
Was CRISPR delivered to zygotes or adult tissue in step 3?
Adult tissue
How were CRISPR components delivered in step 3?
By AAV injection into adult muscle
How many guide RNAs and donor DNAs were used in step 3?
1 guide RNA and 0 donor DNAs
What repair pathway was mainly used in step 3?
NHEJ
How can a single CRISPR cut help restore the DMD reading frame in the Δ50 model?
By exon reframing or exon skipping
What is exon reframing?
NHEJ creates a small indel that restores the reading frame of exon 51
What is exon skipping in this context?
Editing disrupts splicing so exon 51 is skipped, allowing downstream exons to rejoin in frame
What was the key protein-level result after treatment in lecture 31?
Dystrophin expression was restored in treated muscle
What big CRISPR ideas from lecture 31 were emphasized in the testable slide?
CRISPR can be used in mice by injecting zygotes or adult tissue, for knock-ins and knockouts, for making mutations and fixing them, and through HDR or NHEJ repair
What kind of spontaneous base change was emphasized in lecture 32?
Cytosine deamination to uracil
What mutation can cytosine deamination eventually produce after DNA replication?
A GC to AT base-pair substitution
How common was this spontaneous cytosine deamination said to be?
About 100 bases per cell per day
Why is GC to AT mutation important medically?
It causes about half of pathogenic base-pair substitutions in humans
What is a DNA base editor?
A CRISPR-based tool that changes one base to another without making a double-strand break
WHere does cas9 come from? What type of Cas9 is used in adenine base editors?
SpCas9 comes from streptococcus pyogenes a pathogenic gram + bac. Cas9 nickase
How is Cas9 nickase different from regular Cas9?
Cas9 nickase cuts only one DNA strand, while regular Cas9 cuts both
What extra enzyme is attached in an adenine base editor?
A deoxyadenosine deaminase
What chemical change does an adenine base editor make first?
Adenine is changed to inosine
Why does inosine matter in base editing?
It is read like guanine
What base-pair change do ABEs ultimately create?
A·T to G·C
Why are ABEs especially useful for disease repair? how do ABE's work
They can make exact reverse mutations. they bind to target, open up genomic DNA, nick non edited strand and deaminate target A in ssDNA bubble (replaced with inosine)- nicking marks the strand as the "bad strand"-signals dna repair enzymes
What is AAV in lecture 32?
Adeno-associated virus used to deliver CRISPR components into mammalian cells
What was the delivery problem with the ABE-nCas9 system?
The gene was too large to fit into one AAV vector
What is an intein?
A protein element that can splice itself out and join the surrounding protein fragments together
How was the oversized ABE system solved?
It was split into N-terminal and C-terminal halves that reassembled by intein protein splicing. covalently connect N to C.
How many genetic components were needed in the split ABE delivery system?
3
What were the 3 needed components in the split ABE system?
One AAV for the N-terminal ABE half, one AAV for the C-terminal ABE half, and one sgRNA expression cassette
What Duchenne mutation was repaired in lecture 32?
Deletion of exon 44
What site was targeted to repair the ΔEx44 mutation?
The splice acceptor site at the start of exon 45
Why target the splice acceptor of exon 45 in ΔEx44 mice?
To alter splicing so the transcript can be restored to a functional reading frame
How was the split ABE system delivered in the ΔEx44 experiment?
By dual AAV injection into muscle
What was the experimental setup for the mouse legs?
Right leg got saline and left leg got the dual AAV ABE system
What counted as DNA-level success in lecture 32 even if a bystander adenine also changed?
Two adenines changed = success
Why was two-adenine editing still considered a success?
Because the key target adenine was edited and the functional repair goal was achieved
What was the protein-level result of successful ΔEx44 editing?
Dystrophin was restored in treated muscle
What is the big picture connecting lectures 31 and 32?
CRISPR in mice can be used both to build disease models and to repair them, using either HDR/NHEJ editing or base editing in zygotes or adult tissues