CRISPR-Gold: New Gold Standard for Therapeutic Genome Editing? by

Fragile X Syndrome (FXS)

demetraeconomopoulos — 2021-12-02 03:10:20 UTC

https://healthjade.com/fragile-x-syndrome/

RESULTS

demetraeconomopoulos — 2021-12-02 03:10:35 UTC

INTRODUCTION

atakizawa — 2021-12-02 03:14:03 UTC

Challenges/Questions to Address in Future Research

atakizawa — 2021-12-02 03:14:57 UTC

Addressing complex diseases (ie multiple gene targets, epigenetic disease)
Long-term effects
Potential toxicity - Can Gold accumulate in the brain?
Local vs systemic distribution
Cell type-specific delivery
Going beyond mouse models
- How do we assess the efficacy of CRISPR-Gold in addressing complex, human-specific behaviours such as language?

METHODS

demetraeconomopoulos — 2021-12-02 03:19:02 UTC

Experiment 1 - Biocompatibility test of CRISPR-Gold in neurons:

ashleytapp — 2021-12-02 03:58:12 UTC

Preface: There is not much known about nonviral gene editing in the brain, therefore, this experiment first examines whether CRISPR-Gold is biocompatible in neurons.

CRISPR-Gold was applied to neurons, and 2 hours later the residual CRISPR-Gold was removed.
A fresh growth medium was added to neurons and left for 10-14 days.
After this time, neurons were patch clamped or stained with SYTOX red to identify dead cells.
Measured the cytotoxicity and impact of CRISPR-Gold in the hippocampal neurons.

Experiment 1 - CRISPR-Gold is BIOCOMPATIBLE!

ashleytapp — 2021-12-02 04:00:35 UTC

No significant difference between control and CRISPR-Gold-treated neuron in all experiments...

★ CRISPR-Gold does not adversely affect neuronal membrane health (Fig. b&c) and neuronal excitability (Fig. d&e).

★ CRISPR-Gold does not cause cytotoxicity! (Fig. f)

Experiment 2 - CRISPR-Gold-mediated gene editing in brains of Thy1-YFP mice

ashleytapp — 2021-12-02 04:06:31 UTC

Used a Thy1-YFP mouse model: a transgenic mouse model that only expresses yellow fluorescent (YFP) protein in neurons!
Designed sgRNAs and CRISPR RNA to target 5' region of YFP to make indel mutations.
Stereotaxically injected CRISPR-Gold loaded with the Cas9/Cpf1 into the hippocampal dentate gyrus of adult mice!
Watch to see if it could successfuly deliver Cas9 ribonucleotides into the brain.

Experiment 3 - CRISPR-Gold mediated gene editing in the brain of Ai9 mice

ashleytapp — 2021-12-02 04:24:40 UTC

Utilized a Ai9 mouse model: Genetically engineered Ai9 mice express tdTomato fluorescence after Cre-recombination occurs!
Before Cre-recombination, tdTomato gene is silenced due to a lox-P flanked stop codon.
Designed sgRNAs and CRISPR-RNAs to target the ends of the stop codon to remove it = fluorescence occurs.
Cas9 or Cpf1 was injected stereotaxically into hippocampus of Ai9 mice. Fluorescence was monitored by fluorescence histology.

HMB360 Presentation!

ashleytapp — 2021-12-02 04:31:08 UTC

Experiment 4 - CRISPR–Gold-mediated gene editing in multiple cell types

ashleytapp — 2021-12-02 04:58:11 UTC

Preface: Glial cell disfunction causes multiple brain disorders, thus editing glial cells is of great interest.

Utilizing Ai9 mice to identify the cell types in the brain that Cas9 or Cpf1 can edit.
Stained sections of Ai9 mice brains with various cell markers: glial fibrillary acidic protein (GFAP), neuronal nuclear protein (NeuN) and ionized calcium-binding adapter molecule 1 (IBA1).
Histology was used to identify neuronal populations, astrocytes, and microglia.
This was tested in the hippocampus and the striatum.

Experiment 5 - Gene editing in Fmr1 knockout mice with CRISPR–Gold

ashleytapp — 2021-12-02 05:33:58 UTC

Preface: Studies show that excessive mGluR5 signalling causes Fragile X syndrome.

Goal: Knockout mGluR5's encoding gene, Grm5.

Generate CRISPR-Gold sgRNA that targets Grm5.
Stereotaxically inject saline for control group, or CRISPR-Gold into striatum of wild type mice or Fmr1 knockout mice.
Examine whether it is possible to knock down mGluR5 gene in vivo.

Experiment 6 - Behavioural rescue in Fmr1 knockout mice by CRISPR-Gold

ashleytapp — 2021-12-02 05:53:11 UTC

Preface: Mice with FSX commonly have repetitive behaviours.

Goal: Determine if knocking out mGluR5 will rescue these behaviours.

Locomotive activities of the mice were assessed!
Tests = line crossings, rotarod performance, marble burrying, and distance travelled in cage.

Marble burying assay - locomotive test

ashleytapp — 2021-12-02 05:57:34 UTC

Genetics of Fragile X Syndrome

demetraeconomopoulos — 2021-12-02 18:44:05 UTC

https://www.nature.com/articles/ejhg200861

Treatment vs Cure

demetraeconomopoulos — 2021-12-02 18:47:39 UTC

Current treatment: pharmaceuticals such as antidepressants and antipsychotics, which treat symptoms rather than cause. A treatment of the cause is needed.
Enter CRISPR: CRISPR is analogous to a pair of molecular scissors and has the potential to treat the root cause of FXS

demetraeconomopoulos — 2021-12-02 20:43:30 UTC

Goal: Deliver CRISPR Into the Brains of Adult Mice

demetraeconomopoulos — 2021-12-02 21:09:36 UTC

Neurons and other brain cells are notoriously difficult to target, largely due to the blood brain barrier, toxic effects, and because neurons are post-mitotic (they do not divide!)
Delivery vehicles can be divided into viral and non-viral vectors

Advantages and Disadvantages of Non-Viral Delivery

demetraeconomopoulos — 2021-12-03 01:46:46 UTC

Advantages to harness:

Lower immunogenicity
Cost-effectiveness
Availability
Carrying capacity

Challenges to overcome:

Transduction Efficiency
Access to the brain
Applicability to post-mitotic cells

demetraeconomopoulos — 2021-12-03 01:49:57 UTC

Enter: CRISPR-Gold

demetraeconomopoulos — 2021-12-03 01:52:35 UTC

What Gaps Were the Authors Trying to Address using the Gold nanoparticle?

demetraeconomopoulos — 2021-12-03 01:57:06 UTC

Only one previous report of non-viral genome modification in the brain.
CRISPR-Gold was new and had not been used in the brain.
No previous attempt to genetically modify using an RNA-guided endonuclease other than Cas9 (ie Cpf1).

The author's contribution lies in filling these gaps while creating an effective treatment to rescue the phenotype of mice with Autism associated behaviours

DISCUSSION

demetraeconomopoulos — 2021-12-03 02:58:16 UTC

Highlights

demetraeconomopoulos — 2021-12-03 03:11:56 UTC

CRISPR-Gold can deliver both Cas9 and Cpf1 ribonucleoproteins into the brains of adult mice safely!
Effectively editing all major cell types in the brain (microglia, astrocytes, neurons) and resulting in reduced autism-associated behaviours in mice!

Significance

demetraeconomopoulos — 2021-12-03 03:15:15 UTC

This was a comprehensive study that tackled gaps in the scientific literature resulting in statistically significant genotypic and phenotypic rescue without detectable harmful effects.

Strengths

demetraeconomopoulos — 2021-12-03 03:17:59 UTC

Tested both in vitro and in vivo.
Thorough biocompatibility analysis.
Mouse models selected with care.
Non-neuronal cells not overlooked (e.g. glial cells)!
Multiple portions of the brain assessed (e.g. hippocampus, striatum).
Analysis of both genotypic and phenotypic outcomes
Used both Cas9 and Cpf1.
Tested for confounding variables ( e.g. locomotor activities).

Weaknesses

demetraeconomopoulos — 2021-12-03 03:19:08 UTC

Elimination kinetics of GNP remains unknown (potential source of toxicity).
Cell type-specific delivery not achieved.
CRISPR-Gold only acted within a restricted area around the injection site.

demetraeconomopoulos — 2021-12-03 03:22:26 UTC

Experiment 4 - CRISPR-Gold-mediated gene editing in multiple cell types in the brain possible!

atakizawa — 2021-12-03 05:42:20 UTC

Gene edited TdTomato+ cells by Cas9 and Cpf1 were composed of all of three cells types!

★Astrocytes, microgila, neurons (major cell types of the brain) were edited by CRISPR-Gold-mediated Cas9 and Cpf1!

Experiment 2- CRISPR-Gold can deliver Cas9 and Cpf1 in the brain!

atakizawa — 2021-12-03 05:43:48 UTC

In both Cas9 and Cpf1, YFP expression level was significantly decreased!

Indel mutation was successfully induced by CRISPR-Gold-mediated Cas9 and Cpf1!

★ CRISPR-Gold delivered Cas9 and Cpf1 in the brain!

Experiment 6 - CRISPR-Gold rescued repetitive behaviours of FXS!

atakizawa — 2021-12-03 05:46:35 UTC

Marble burring assay: no significant difference after control vs mGluR5-CRISPR injection in wild type mice ↔︎ Significant decrease in the percentage of marble buried in Fmr1 KO mice.

Line crossing: No significant difference in number of jumps in WT. ↔︎ Significant drop in jumping behaviour in Fmr1 KO mice.

★ Excessive digging/jumping behaviours are rescued by CRISPR-Gold-mediated mGluR5 injection in a FXS model of a mouse!

Empty cage observation: The number of line crossing increased in Fmr1 KO mice compare to WT as it is the phenotype of FXS. However, mGluR5-CRISPR did not decrease the number of line crossing.

★ Exaggerated mGluR5 signalling is not involved in the hyperlocomotor activity in FXS.

Experiment 3 - CRISPR-Gold-mediated gene editing in targeted DNA gene!

atakizawa — 2021-12-03 05:47:09 UTC

TdTomato expression after the injection of CRISPR-Gold-mediated Cas9 and Cpf1 significantly increased!

Both Cas9 and Cpf1 removed stop codon to induce TdTomato expression.

★ CRISPR-Gold-mediated Cas9 and Cpf1 could successfully edit targeted gene in the hippocampus!

Experiment 5 - Successful gene editing in FXS mouse model!

atakizawa — 2021-12-03 05:47:59 UTC

A decrease of both Grm5 and mGluR5 protein expression level after the injection of CRISPR-Gold-mediated-mGluR5 in Fmr1 knockout mice!

★ A successful gene editing of Grm5 (potential cause of FXS) by CRISPR-Gold-mediated-mGluR5 in a FXS mouse model!

demetraeconomopoulos — 2021-12-03 19:14:04 UTC

demetraeconomopoulos — 2021-12-03 19:18:29 UTC

CRISPR-Gold Research: A Timeline

demetraeconomopoulos — 2021-12-03 19:23:01 UTC

demetraeconomopoulos — 2021-12-03 19:27:50 UTC

demetraeconomopoulos — 2021-12-03 19:32:52 UTC

As a polymetric nanoparticle, CRISPR-Gold excels in efficient delivery but falls short when it comes to prolonged tissue deposition.

Suggestions for improvement include biodegradable design for tissue deposition and magnetic nanoparticles for cell/tissue specificity.

OUTLOOK

demetraeconomopoulos — 2021-12-03 22:35:35 UTC

The Future of CRISPR-Gold - What Have Lee and colleagues Been up to Since Publishing Their Paper?

demetraeconomopoulos — 2021-12-03 22:55:41 UTC

With newfound funding at their disposal from investors, they have developed NanoGalaxy™, a library spanning thousands of unique non-viral, non-lipid, polymer nanoparticles! They have expressed hopes of bringing this library to the clinic, to overcome the challenge of delivery for gene therapy in a wide range of diseases.

CRISPR-Gold in 2021

demetraeconomopoulos — 2021-12-03 23:15:00 UTC

A search for articles about "CRISPR-Gold" in the UofT library yields a mere 2 pages worth of results when filtered to include publications from 2021. While review papers continue to cite CRISPR-Gold, primary research has died down significantly. It seems that CRISPR-Gold and the research done by Lee et al was a stepping stone to Genedit's new, comprehensive system. However, CRISPR-Gold is undeniably promising and Lee et al's work remains impactful in 2021. The future is unknown, and there are more questions to answer about CRISPR-Gold!

demetraeconomopoulos — 2021-12-03 23:20:19 UTC

Acknowledgement

demetraeconomopoulos — 2021-12-03 23:45:27 UTC

Demetra Economopoulos: Title, Introduction, Discussion, Outlook
Ashley Tapp: Methods, References + formatting
Ayu Takizawa: Results, Challenge/Questions to Address in Future

Reference list

ashleytapp — 2021-12-04 00:05:15 UTC

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Mandal, A. (2018). What is Fragile X Syndrome?. Retrieved 1 December 2021, from https://www.news-medical.net/health/What-is-Fragile-X-Syndrome.aspx

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PPM. (2015). Glial cells activated [Image]. Retrieved from https://www.practicalpainmanagement.com/pain/other/glial-cell-activation-neuroinflammation-how-they-cause-centralized-pain

Saey, T. H. (2020). CRISPR enters its first human clinical trials. Science News. Retrieved December 4, 2021, from https://www.sciencenews.org/article/crispr-gene-editor-first-human-clinical-trials.

Shahbazi, R., Sghia-Hughes, G., Reid, J., Kubek, S., Haworth, K., & Humbert, O. et al. (2019). Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations. Nature Materials, 18(10), 1124-1132. doi: 10.1038/s41563-019-0385-5

Trenkmann, M. (2018). Gold rush to gene-editing in the brain. Nature Reviews Genetics, 19(9), 532-533. doi: 10.1038/s41576-018-0038-6

Youtube - UC Berkeley. (2018). Research towards a treatment for autism [Video]. Retrieved from https://www.youtube.com/watch?v=KJaW9-7BcVs&t=12s&ab_channel=UCBerkeley