RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA–guide RNA complex residing at the 5′ of the crRNA. The activity of Cas13a was maximal for guide RNAs 24–30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70–76%), mCherry fluorescence (72%), and EML4-ALK at the protein (>80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.