• Robust APP by Crispr/Cas9 editing was seen in vitro and in vivo mice models and iPSC-derived Alzheimer’s neurons with no off-target effects, with more than 80% accuracy in AAV9-sgRNA delivery.
  

• CRISPR/Cas9 extreme C-terminus editing did not affect neurophysiology including synapse function, neurite growth, and spine density.

• Shows proof for selective APP cleavage silencing strategy, which has future implications for therapeutic interventions to prevent Alzheimer’s Disease using CRISPR technology.

• Though the authors state no off-target effects, they only tested a few spots they believed could be off-targeted (Hind3 and Sac2) using PCR.

  • The authors state that their off-target analysis cannot detect small double-stranded breaks in DNA and that single nucleotide analysis is needed. Instead, a bulk RNAseq study could be used to see which genes experience off-target effects, or single-cell RNA sequencing like they suggest in the article because the effects of cutting could elsewhere, and not only occur around the protein⁵.

• Though C-terminus truncation was done in iPSC cells with a London mutation, an Alzheimer’s Disease familial mutation that increases the production of AB, the researchers did not use an in vivo Alzheimer’s Disease model⁶. 

  • The authors state that they would need a future study to conclude whether editing the C-terminus can prevent amyloid beta buildup by silencing the B-cleavage pathway⁷. 
    

• No conclusion of whether the editing C-terminus would stay consistent over a long period, and if the neuronal physiological function of the C and N terminus stays untouched.

  • The authors state that they would need to complete a longitudinal study in vivo and in vitro to know whether neuronal physiological function degrades over time.
    

• Lack of behavioural testing in live mice after C-terminus cleavage

  • Though no genetic off-target effects were found, there was no motor test to conclude whether C-terminus truncation changed mice's physical behaviour.

This article shows conceptual proof that editing the C-terminus using CRISPR/Cas9 changes the balance of APP cleavage and increases neuroprotective a-cleavage. The authors propose that their system works by editing the YENPTY motif that initiates the internalization of APP in endosomes, which causes BACE-1 (b-secretase) to cut APP into amyloid beta protein⁸. By editing, they attenuate YENPTY’s function and amyloid precursor protein stays on the surface where a-cleavage takes place, and they presume this is why a-cleavage is unregulated. In mice, they found that APP homologues take over in the absence of APP B-cleavage⁹. The in vivo study in newborn mice shows that there may be a sensitivity period in which the AAV is permeable to the brain by intracerebroventricular injection. Therefore, in theory, a fetus’ that is screened for a familial genetic mutation for Alzheimer’s, can be administered an AAV with a CRISPR Cas/9 to truncate the C-terminus and stop any pathological symptoms that follow Alzheimer’s disease¹⁰. Lastly, C-terminus editing did not affect the N-terminus of APP, and they propose that the N-terminus has a physiological role in axon growth and neuronal differentiation. By far, the largest limitations of this article include the lack of using an in vivo Alzheimer’s mouse model with the London mutation and the lack of a longitudinal study. In an article where the authors use transgenic mice with the human version of the Swedish mutation, in the amyloid precursor protein (APPswe), and introduced indel mutations to APP by AAV-sgRNA technology, there showed decreased AB levels by 60%¹¹. Another study also used the Swedish mutation and one AVV-sgRNA intrahippocampal injection which alleviated AB plaques, by introducing a point mutation, for up to 6 months in live mice, and ameliorated microgliosis, neurite dystrophy, and cognitive impairment¹². More tests would have to be done using the London mutation in APP to see if C-terminus truncation therapy can reverse AB buildup in an Alzheimer’s model and alleviate the symptoms like these two other studies show and have long-lasting effects as well.

Subsequent repair of these DNA breaks is naturally error-prone, resulting in insertions and deletions (indels) at the cut sites. These indels disrupt the translational reading frame and effectively truncate the APP protein¹. By editing the C-terminus of APP, the CRISPR/Cas9 strategy attenuates β-cleavage, which is the cleavage of APP by β-secretases that leads to the production of amyloid beta (Aβ)^2,3. At the same time, it promotes α-cleavage, which is the cleavage of APP by α-secretases and is thought to be neuroprotective.

The mechanism of action of this strategy likely involves limiting the interaction between APP and β-secretase (BACE-1), the enzyme responsible for β-cleavage. By editing the C-terminus of APP, the proximity between APP and BACE-1 is reduced, leading to a decrease in β-cleavage and Aβ production³. Additionally, the retention of APP at the plasma membrane may facilitate the upregulation of α-cleavage.

In the context study, Sun et al, the researchers focused on the amyloid precursor protein (APP), which is normally present in the brain^2,4. APP can be cleaved by enzymes called β-secretases and γ-secretases to generate Aβ, the main component of Aβ plaques. This is known as the amyloidogenic pathway. Alternatively, APP can be cleaved by α-secretases, which is the non-amyloidogenic pathway and is thought to be neuroprotective. 

Residues T668 and YY682-Y687 in the C-terminus play a role in AB production where APP phosphorylated or turned on at t668 colocalize with BACE-1 in endoscopes which is why the BIfC method was used to approximate how close APP phosphorylated at T668 and BACE-1 in endosomes come close together and cleavage of APP happens⁴.

The researchers used a CRISPR/Cas9 system to edit the APP C-terminus in cell and animal models. They found that editing the C-terminus resulted in reduced β-cleavage and Aβ production while promoting α-cleavage.⁴ Importantly, the editing did not have deleterious effects on neurophysiology in vitro. The researchers also observed the editing of endogenous APP in mouse brains with no detectable off-target effects.⁴

The study provides proof-of-concept for a selective APP silencing strategy using CRISPR/Cas9 gene editing. By manipulating the amyloid pathway, this approach has the potential to attenuate the pathological effects of Aβ in neurodegenerative diseases like Alzheimer's. However, further studies are needed to understand the long-term effects and to test the strategy in an Alzheimer's disease mouse model.

1. Implant transgenic IPSC-derived neurons from a patient with London mutation into a paternal mouse and cross it with a maternal mouse without the mutation. Since the mutation is autosomal dominant, at least one of their offspring should have it.

2. Have a group of wild-type mice and London mutation mice and deliver the AAV-sgRNA Cas9 vector to truncate the C-terminus of Amyloid Beta Protein in neonatal mice, with the mutation and to the wild type, just a regular AAV-Cas9 vector.

3. Perform behavioural tasks including spatial, cognitive and physical tests to see if C-terminus truncation changes normal mice behaviour. The tasks could be a Y-maze, open field test, and the rotarod test, respectfully.

4. Mice mature to full adults after about 7 weeks, so an Elisa assay, by taking a sample of the mice's brain tissue, can be done to see if amyloid beta protein (AB40/42) levels are low, which would assume less amyloid beta plaque build-up in the brain of the mice.  

Expected Results:

Lack of amyloid beta build-up in the brain, the truncation of the C-terminus can be used as a therapeutic approach to prevent familial Alzheimer’s Disease and its injection is persistent for almost 2 months. This study would address whether adverse behavioural function occurs due to C-truncation, and if C-terminus truncation when delivered early enough can prevent normal disease progression. More testing, including a BiCF test, needs to be done between a-secretase and APP with the extreme C-terminus truncated, to understand the mechanism of a-cleavage with respect to this CRISPR technique upregulating it.