1. Assessing the role of the ALS-associated gene NEK1 in zebrafish motor neuron development
Amy Stark

2. What is Amyotrophic Lateral Sclerosis?
Commonly known as ALS or Lou Gehrig’s disease, it is a neurodegenerative disease characterized by the progressive death of motor neurons in the brain and spinal cord responsible for voluntary muscle movement.
5-10% of ALS cases are hereditary

3. Effects of the disease
Characterized by weakening and paralysis of muscles involved in arms, legs, hands, swallowing, or speech
Begins as mild symptoms, then spreads
Respiratory system becomes affected
Death usually within 3-5 years of onset
Most typical cause of death is respiratory failure

4. NEK1 Discovery in 2016 was funded by the ALS Ice Bucket Challenge
Some known functions in regulating meiosis and repairing DNA damage
Loss-of-function variants linked to 3% of ALS cases
The p.Arg261His amino acid substitution could be a particular mutation linked to the disease
Normal function???
“Focusing on one of the genes and one of the mutations
Brain (2016) 139 (5): e28.

5. Two methods to study ALS
General mutations in NEK1
Use CRISPR/Cas9 system to introduce mutations into the gene in zebrafish
Mutations should cause loss of function of the gene
Analyze the zebrafish to see motor neuron development issues
Point mutation to simulate human ALS
Introduce the p.Arg261His amino acid substitution in zebrafish
Observe for motor neuron defects
Part 2 is to compare the human mutation to a general mutation
“Model human disease”

6. CRISPR/Cas9 genome editing
Clustered regularly interspaced short palindromic repeats
Cut some or insert some bases to cause mutation
Part two only adds in the oligonucleotide region we want
Injects a DNA strand to serve as a repair pathway that includes the new amino acid
FIND A NEW FIGUREEEEEE

7. Using the CRISPRs Part 1 Cas9 protein Guide RNA Part 2 Cas9 protein
Oligonucleotide with CAC sequence
First part:
Only Cas9 and guide RNA to just any mutation
Second part:
Cas9 and guide RNA and oligonucleotide containing CAC sequence

8. CRISPR gRNA design Arginine amino acid 50 base pairs downstream
Cut at GCT exon 8
Inject it to try to make mutation
Didn’t work
2. Injecting guide RNA and the oligonucleotide that contains the histidine to change
Arginine amino acid
50 base pairs downstream

9. Zebrafish motor neurons with GFP
Phenotypic Analysis
Swimming patterns
Observe growing fish for any abnormal swimming abilities
GFP results
Fluorescent protein used to highlight motor neurons in zebrafish
Observe motor neuron shortening or growth defects
Zebrafish motor neurons with GFP

10. Conclusions
So far, CRISPR has not successfully introduced a mutation because double-stranded cuts aren’t being made
Once we get a CRISPR to cut and cause a mutation, analyze effects in zebrafish
Inject oligonucleotide with CAC sequence for specific amino acid substitution to simulate human ALS

11. Acknowledgements The Dr. Jeffrey Trimarchi Laboratory
Rebecca Chowdhury
Lauren Laboissonniere