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Although utilization of CRISPR-Cas9 in AD study has merely started, CRISPR is a perfect tool to analyze gene disruption and the role of that gene in AD progression⁴. The biggest problem for CRISPR-Cas9 to become a therapeutic tool is the off-targets effects⁴. Nevertheless, new strategies including biased and unbiased off-targets detection methods, guide RNA Modification and Engineering, improved Cas Variants are emerging to help minimize the off-target effects⁶. CRISPR/Cas9 based therapies can be expected to get approved for AD treatment in the near future.

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CRISPR/Cas9: genome editing tool that allows manipulation of the DNA sequence.

Components:
1. “Molecular scissors” that cut DNA at specific sites²
2. Guide RNA that ensures the correct cutting sites of Cas9 enzyme²

Process:
1. Locate a specific site in the DNA with guide RNA
2. Cas9 enzyme cuts that specific site of the DNA
3. Modify the DNA
4. DNA undergoes a repair process²

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• Quantities of Genes associated with AD are extensive. CRISPR/Cas9 editing allows scientists to apply large scale modeling by generating multiple single-nucleotide variants per gene across different targets⁴.

• CRISPR/Cas9 can help find out new proteins for AD⁴. Through utilization of CRISPR/Cas9 system, experts from University of Tokyo identified a new protein, calcium and integrin-binding protein 1⁴, which is associated with the abnormally high amyloid-beta levels⁵.

• CRISPR/Cas9 can be used to modify mutations in presenilin genes⁴. As mentioned in the article, mutations in PSEN2 can be one of the causes of AD¹. It was demonstrated by previous studies that patients with high amyloid-beta 42/40 ratio in basal forebrain cholinergic neurons, which is the first types of cells affected in AD, displayed electrophysiological deficiency⁴. Through correction of PSEN2 mutation with CRISPR/Cas9, scientist can effectively restore the electrophysiological function⁴.

• CRISPR/Cas9 can be used to activate genetic variants associated with amyloid-beta level reduction, which may potentially prevent the progress of AD⁴. A genetic variant named A673T was discovered to reduce the probability of catching AD by a factor of four⁴. With CRISPR/Cas9 system, researchers from Laval University were able to activate the A673T variant in petri dish studies⁴.

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Demands for novel therapies against AD continue to raise as traditional AD treatment merely provide patients with symptomatic relief⁴. Currently, it is known that accumulation of amyloid-beta peptide in the brain can cause AD. Therefore, if the amyloid-beta levels are in control, pathology can be slowed down. Immunotherapy studies focused on the toxic amyloid-beta species were shown to be promising¹. In this study, the authors came up with an experiment to prove the feasibility of reducing amyloid-beta levels by disrupting the APP mutation with CRISPR/Cas9 system. The success of this study paves the way for potential AD treatment approach with CRISPR/Cas9 gene editing. There are many other ways in which CRISPR/Cas9 can be harnessed to tackle AD.

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Future studies can focus on increasing the efficacy of using the CRISPR/Cas9 system to target in vivo models. For instance, ways to improve the previous in vivo models, how much indel formation observed is going to be considered significant, and whether or not it can successfully reduce amyloid-beta levels in model organisms and decrease pathogenesis of the disease.

Inject AVV-mediated CRISPR/Cas9 multilaterally, more CRISPR/Cas9 expressing cells.

Test statistical significance using the P-value.

Detect Amyloid-beta levels in the brain via positron emission tomography (PET) scans.

Determine whether there is any molecular or cognitive dysfunction in different treated animal models. E.g. Check for symptoms of spatial memory impairment in mice treated with in vivo injections of control and experimental designs. Accumulation of amyloid-beta levels in the brain can result in memory deficit⁷.

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CRISPR/Cas9 with other neurodegenerative diseases

Use CRISPR/Cas9 to knock out trinucleotide-repeats of exon 1 in mice with Huntington⁸.

successfully decreased brain pathology and motor deficiency⁸.

Did not impact the viability of the mice⁸.

Use CRISPR/Cas9 to target mutant alleles of SNPs in mice with Huntington's disease⁹.

see a decrease in mutant huntingtin mRNA and protein level⁹.

CRISPR/Cas9 and other targets of AD

Target APOE4 (apolipoprotein E4) allele using CRISPR/Cas9¹⁰

APOE4 increases the risk of late-onset AD, but its isoforms APOE2, and APOE3 do not¹⁰

converting APOE4 to APOE2 or APOE3 using CRISPR/Cas9 decreases the AD pathologies¹⁰

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in vivo injections were unilateral as opposed to multilateral
- more injection can lead to higher indel formation
- In vivo results were not thoroughly investigated.

Effect on pathogenesis is not discussed
- How the in vivo experiments relates to the pathogenesis is unknown
-  How much indel formation is significant should be studied

Small sample size
- 3 samples from the APPswe and 2 samples from the control
- More of a proof of concept experiment

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Focused on the source rather than outcome
- Targeted at the genetic level
- Prior studies that target amyloid-beta proteins

Target only the mutant allele
- gRNAs only targetted mutant allele
- Wild-type allele was unaffected
- Leads to fewer side effects

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Considerations for designing gRNAs
- Specificity of gRNAs
- Length of the nucleotide of guide RNAs

A good therapeutic approach is dependent on a number of elements. Slight alternation can increase or decrease efficiency.

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Rationale: Disruption of the mutant allele can inhibit the overproduction of amyloid-beta in patient’s cells¹.

CRISPR/Cas9 = selectively target/disrupt APPSW

• Designed guide RNAs to target both the mutant allele and the wild type allele¹

• 3 Guide RNAs: 20 nucleotide (NT), 19 NT, 17 NT = test efficiency and specificity of genome editing¹

• Performed targeted deep sequencing with CRISPR/ Cas9 system to examine the extent of genome editing¹

• After having the guide specificity confirmed with another targeted deep sequencing, the authors determined the locus of the indels¹

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Filip Group 4
Chiaki Shuzenji, Shichun Xu, Le Chen

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• Amyloid plaque = indicator for AD
--> formed by the accumulation of the amyloid-beta which is generated at the APP cleavage site¹.

• APP swe (mutation that originated from Sweden) = high amyloid-beta levels through increasing beta-secretase cleavage activity¹.

• Heterozygous for APPswe = 3x more susceptible to abnormal amyloid-beta levels¹.

• DNA sequences with certain PAM sites can be targeted with short gRNA at 20- nucleotides-long¹.

• Both NHEJ and HDR are involved in the repair pathways which occurs when 5’ end of the PAM sites breaks¹.

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1.    György, B., Lööv, C., Zaborowski, M., Takeda, S., Kleinstiver, B., Commins, C., Kastanenka, K., Mu, D., Volak, A., Giedraitis, V., Lannfelt, L., Maguire, C., Joung, J., Hyman, B., Breakefield, X. and Ingelsson, M., 2018. CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease. Molecular Therapy - Nucleic Acids, 11, pp.429-440.

2.    Yourgenome.org. 2021. What is CRISPR-Cas9?. [online] Available at: <https://www.yourgenome.org/facts/what-is-crispr-cas9> [Accessed 3 December 2021].

3.  Youtube.com. 2021. What is Alzheimer's disease? - Ivan Seah Yu Jun. [online] Available at: <https://www.youtube.com/watch?v=yJXTXN4xrI8> [Accessed 3 December 2021].

4.  Synthego.com. 2021. Synthego | Full Stack Genome Engineering. [online] Available at: <https://www.synthego.com/blog/alzheimers-crispr> [Accessed 3 December 2021].

5. ScienceDaily. 2021. CRISPR helps identify potential Alzheimer's-related protein. [online] Available at: <https://www.sciencedaily.com/releases/2020/04/200421112529.htm> [Accessed 3 December 2021].

6. Naeem, M., Majeed, S., Hoque, M. and Ahmad, I., 2020. Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing. Cells, 9(7), p.1608. [online] Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407193/ [Accessed 3 December 2021].

7. Faucher, P., Mons, N., Micheau, J., Louis, C. and Beracochea, D., 2016. Hippocampal Injections of Oligomeric Amyloid β-peptide (1–42) Induce Selective Working Memory Deficits and Long-lasting Alterations of ERK Signaling Pathway. Frontiers in Aging Neuroscience, 7. [online] Available at: https://www.frontiersin.org/articles/10.3389/fnagi.2015.00245/full [Accessed 3 December 2021].

8. Yang, S., Chang, R., Yang, H., Zhao, T., Hong, Y., Kong, H., Sun, X., Qin, Z., Jin, P., Li, S. and Li, X., 2017. CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease. Journal of Clinical Investigation, 127(7), pp.2719-2724.

[online] Available at:  https://www.readcube.com/articles/10.1172%2Fjci92087 [Accessed 3 December 2021].

9. Monteys, A., Ebanks, S., Keiser, M. and Davidson, B., 2017. CRISPR/Cas9 Editing of the Mutant Huntingtin Allele In Vitro and In Vivo. Molecular Therapy, 25(1), pp.12-23.

[online] Available at: https://www.researchgate.net/publication/312103582_CRISPRCas9_Editing_of_the_Mutant_Huntingtin_Allele_In_Vitro_and_In_Vivo [Accessed 3 December 2021].

10. Lin YT, Seo J, Gao F, et al. APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types [published correction appears in Neuron. 2018 Jun 27;98 (6):1294]. Neuron. 2018;98(6):1141-1154

[online] Available at: https://www.sciencedirect.com/science/article/pii/S0896627318303805 [Accessed 3 December 2021].

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Introduction: Chiaki Shuzenji & Le Chen
Method: Chiaki Shuzenji & Le Chen
Results: Shichun Xu
Discussion: Shichun Xu & Chiaki Shuzenji
Outlook: Shichun Xu & Le Chen

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- 20-NT (SW1) and 19-NT (SW2) gRNAs are most effective at targeting the mutant allele and lowering A-beta levels1

- The 20-NT wild-type gRNA is efficient at targeting the non-mutant allele and lowering A-beta levels1

= future gRNA designs should at least exceed 17-NT for gRNAs to be efficient at targeting their site of interest.

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- After 4-8 weeks post-injection of AAV9-Cas9 and AAV9 SW1-gRNA in transgenic mice, indel formation occurred in the APPswe allele of the hippocampus, not in the non-injected cerebellum¹.

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in vivo CRISPR/Cas9 indels in Tg2576 mice

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in vivo CRISPR/Cas9 indels in Tg2576 mice

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- ~2% indel formation is detected in AAV-Cas9 SW1-gRNAs¹.
- No indel formation in cells treated with the empty vector (EV)¹.

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- 6-fold increase in amyloid-beta levels in mutant cell lines compared to the wild-type cell line¹.
- SW3 gRNAs did not have a significant effect on the reduction of amyloid-beta levels¹.

Significance: the result from targeting cell lines with the CRISPR/Cas9 system is successful, as seen by the resulting amyloid-beta levels.

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Amyloid-Beta levels from mutant and wild-type cell line.

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Amyloid-Beta levels from mutant and wild-type cell line.

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- 50-60% decrease in amyloid-beta levels in SW1 and SW2 gRNAS, no reduction was seen in SW3 gRNAs¹.
- gRNAs that target the mutant allele did not have an effect on the wild-type cell lines, thus, no reduction in amyloid-beta¹.
- WT gRNAs significantly decreased the levels of amyloid-beta in wild-type cells and in cell lines with the wild-type allele¹.

Significance: SW1 and SW2 gRNAs successfully decreased amyloid-beta levels in mutant cell strains, this was not seen in SW3 gRNAs.

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- SW1 and SW2 gRNAs created indels in the APPsw/wt cell line¹.
- No indel formation in the APP Swedish allele with SW3 and WT gRNAs¹.
- Wild type gRNAs only caused indel formation in the wild type cell lines and the wild type allele of the APP Swedish cell line¹.
- In the APPwt/wt cell line, no indel was seen in either allele with cells treated with SW1, SW2, or SW3 gRNAs¹.

Significance: APP wild type allele or Swedish allele can be specifically targeted using the CRISPR/Cas9 system.

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Adult Tg2576 transgenic mice
Process:
- Adult Tg2576 mice were transduced unilaterally with AAV9-Cas9 and AAV9-gRNA in the hippocampus
2. After 1 - 2 months, the mice were humanely sacrificed
3. The treated hippocampus and control cerebellum were separated and analyzed using genomic DNA sequencing

Embryonic day 14 (E14) Tg2576 mice
- E14 Tg2576 mice were used to create primary cortical neuronal cultures
- The cultures were transduced with AAV1-Cas9 and AAV1-gRNA in vivo
- After 21 days in vivo, the cells were collected and sequenced

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Fibroblasts from human APPswe carriers and their wild type relatives were used

Process:
1. The cells were then transfected with S.pyogenes Cas9-2A-GFP and gRNAs
2. The successfully transfected cells are sorted using the fluorescence cell sorter (FACS)
3. The successfully transfected cells were analyzed:
- sequencing
- western blot for the intracellular APP levels
- extracellular secretion of amyloid-beta40 and 42¹

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- Determine how CRISPR-Cas9 mediated disruption of APPswe mutation alters Aꞵ formation¹

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- mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1), presenilin 2 (PSEN2)¹

- APP mutation leads to amyloid-beta (Aꞵ) accumulation
--> formation of Aꞵ plaque¹

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A neurodegenerative disorder
- characterized by memory loss, cognitive impairments³

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What is Alzheimer's disease? - Ivan Seah Yu Jun   Reference #3                               Obtained from https://www.youtube.com/watch?v=yJXTXN4xrI8

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What is Alzheimer's disease? - Ivan Seah Yu Jun