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

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 http://www.alsfoundation.org/learn/

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

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.

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”

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 http://www.frontiersin.org/files/Articles/162559/fgene-06-00300-HTML/image_m/fgene-06-00300-g001.jpg

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 http://www.nature.com/article-assets/npg/srep/2015/150908/srep13745/images_hires/w926/srep13745-f1.jpg

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

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

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

Acknowledgements The Dr. Jeffrey Trimarchi Laboratory Rebecca Chowdhury Lauren Laboissonniere