Amyotrophic Lateral Sclerosis (ALS) is a rapid progressive degenerative disease of motor neurons that control muscles of limbs, tongue, and lungs. Unfortunately, this destructive disease still remains untreatable largely due to a limited understanding of disease mechanisms and the absence of a suitable model organism for drug screening. Currently, known causative genetic mutations only account for ~6% of ALS cases despite the total ALS heritability being 50%. Not only is there a tremendous knowledge gap in the genetic basis of ALS, but also the associated clinical phenotypes are complicated and disconnected to the known genetics. Therefore, the goal of our study is to identify and characterize the genetic factors underlying the disease and to specifically correlate these alterations to various groups of ALS clinical features. To understand genetic mechanism underlying ALS, we have recruited familial ALS (fALS) patients, sporadic ALS (sALS) patients, and sALS trios consisting of ALS patient and their unaffected parents. Consented patient samples were subjected to exome-sequencing, whole-genome sequencing, and SNP array analysis. As a result, we identified, in addition to rare damaging variants in ALS causative genes such as SOD1 and TDP43, de-novo compound heterozygous mutations in cytoskeletal-related proteins such as DNAH2, DYNC2LI1, STARD9 and NRP2. To functionally validate candidate genes, CRISPR knockout technology was performed in mouse embryonic stem cells (mESC) and the zebrafish model system. Two CRISPR targets per gene were generated and used for the knockout to allow for easy validation tests with PCR and RT-PCR. At present, homozygous knockout mouse embryonic stem cells and heterozygous knockout F1 zebrafish are available. Crosses are underway to produce homozygous knockout F2 embryos. In parallel, ex vivo differentiations of mESC are in progress to compare neurite outgrowth of motor neurons between the knockout and the wildtype. Preliminary results of NRP2-CRISPR injected embryos demonstrate a bent-body phenotype, resembling the phenotype of ALS zebrafish. DNAH2-, DYNC2LI1-, and STARD9-CRISPR injected embryos also showed slight locomotive defects. Taken together, our results suggest that dysregulation of cytoskeletal-related proteins could play an important role in ALS disease mechanism. Contributing to the field, we have identified probable causing genes of ALS, and provided cellular and whole-organism ALS models that are pathway-specific for specialized ALS drug screening.