Finally, performing NGS analysis, they found M146L gRNA to be specific to the mutatioj site reducing the off-target effect. Since this treatment showed promising results, they concluded that it could be developed as a potential therapy for early onset AD.

Then they used fluorescence resonance energy transfer/fluorescence lifetime imaging (FRET/FLIM) and found that the conformational changes in PS1 that's caused by disrupting PSEN1 M146L using CRISPR-Cas9 is the reason for Aβ42/40 ratio reduction.

Then they checked the Aβ42/40 ratio by treating the samples with CRISPR-Cas9. As a result, there was reduction in the ration but not similar to control. So they concluded that this treatment causes partial reduction in Aβ42/40 ratio.

Then treated the samples with SpCas9 enzyme and gRNA. Sequencing the treated samples revealed that the enzyme disrupted the mutant at NGG PAM site in PSEN1 M146L allele, but not the wild type. This indicates the selectivity of SpCas9 enzyme.

The study obtained human fibroblasts with PSEN1 M146L mutation from 6 people, and 4 controls (2 family members, 2 unrelated). First they sequenced the DNA to check the mutation present.

The cut made by SpCas9 at the mutated site was repaired by NHEJ mechanism. It introduced indel causing frame shift mutation and eventually disrupting the PSEN1 M146L allele. Since PSEN1 wild-type was intact, Aβ42/40 ratio was reduced but did not restore fully compared to control.

The study selected PSEN1 M146L mutated gene since it's autosomal dominant, and disrupting the allele over correcting would still allow the wild-type allele to express. (This is very strategic and interesting. We'll look into the results in upcoming days).

Found the answer (perhaps). It's not early-onset an ideal candidate, but genes mutated in it are autosomal dominant, meaning 1 allele causes the disease. If 2 alleles mutated, both of them need to be edited posing it a challenge. In autosomal dominant genes, we focus only on one allele.

Therefore, the study chose NHEJ acc. to their previous study to disrupt PSEN1 M146L mutation by creating indel mutation. Streptococcus pyogenes Cas9 (SpCas9) is used, as it recognizes 5'-NGG-3' PAM site, making it suitable for A to C (AGG) mutation to be recognized.

Cas9 create double strand break after recognition of PAM site. The break is repairs either through non-homologous end joining (NHEJ), or by homology-directed repair. NHEJ repair is less robust, creates indels, causing frame shift or nonsense mutation. This disrupts the target gene.

The CRISPR-Cas9 system is used to edit genes. Factors to consider while choosing it are the Protospacer adjacent motif (PAM) site and guide RNA specific to the gene of interest. PAM is 2-6 nts specifically chosen for Cas9 recognition. gRNA + Cas9 bind next to PAM in the target strand and make a cut.

The study says that the catalytic effect of the PSEN1 M146L (A > C) mutation in Aβ formation is also observed outside the brain, such as in fibroblasts. It provides easy access to study the mutation, similar to brain samples. In this study, they selected CRISPR-Cas9 suitable for this mutation.

We know the mechanism of Aβ formation (refer image). But the increasing production of Aβ42 over Aβ40 is unknown. As the study suggested, the conformation of the enzymes could play a role. But could there be something else happening? Maybe the mutations in PS1 recruit other enzymes here. #opinion

It seems the reason for increase in Aβ42 over Aβ40 ratio is unclear. But the study speculates that certain conformation of PS1 due to mutations could play a role. Since Aβ42/40 ratio is a biomarker for Alzheimer's-related diseases, it is important to understand the mechanism unders these conditions.

Cont/2

Aβ is formed at the end of the enzymatic processing of amyloid precursor protein (APP) in the amyloidogenic pathway. And Presenilin 1 (PS1) protein, expressed by the PSEN1 gene, cleaves APP to form Aβ. Under pathological conditions, Aβ accumulates to form plaques, a hallmark of AD.

Plot: Alzheimer's disease (AD) is a late-onset (LO) disease. But 5% of young adults (early-onset/EO) are affected too. Previous studies observed symptoms involving certain mutations from LOAD in EO. Particularly, mutations in PSEN1 form amyloid β plaques, increasing the Aβ42/40 ratio (AD biomarker).