Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, and current treatments are ineffective. Reduction of SOD1 is the primary clinical approach to treatment, with several strategies to silence SOD1 expression under preclinical and clinical investigation. Small interfering RNAs (siRNAs) are a new class of drugs for which potent and sustained modulation of gene expression has been demonstrated. The effectiveness of siRNA knockdown is modulated by its chemical modification properties. We evaluated the impact of chemical variance and siRNA length on target knockdown and identified a potent siRNA that modulates SOD1 expression in SOD1-G93A mice. When siRNA compounds are not homologous to a model species, performing preclinical studies is challenging. An alternative strategy to expand targetability against genes across different species is to adapt a potent human-targeting lead siRNA for the model species by changing the nucleotide sequence of the siRNA guide strand to match the target site in the model species. I systematically evaluated this strategy of “animalizing” the guide strand and found that the ability to generate species-active compounds based on human leads was highly affected by the target sequence and number of mismatches. The potency of a mouse-active compound generated from this strategy was significantly lower than a mouse-active compound identified from a focus screen. Although our strategy failed to generate a dog-active compound to target canine SOD1, a canine SOD1 focus screen identified potent siRNAs. Thus ,parallel screening for species-active compounds may be a more uniform strategy for the fast clinical progression of RNAi-based drugs.