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.

Approval of therapeutic RNA molecules, including RNA vaccines, has paved the way for next-generation treatment strategies for various diseases. Oligonucleotide-based therapeutics hold particular promise for treating incurable muscular dystrophies, including Duchenne muscular dystrophy (DMD). DMD is a severe monogenic disease triggered by deletions, duplications, or point mutations in the DMD gene, which encodes a membrane-linked cytoskeletal protein to protect muscle fibers from contraction-induced injury. Patients with DMD inevitably succumb to muscle degeneration and atrophy early in life, leading to premature death from cardiac and respiratory failure. Thus far, the disease has thwarted all curative strategies. Transcriptomic manipulation, employing exon skipping using antisense oligonucleotides (ASO), has made significant progress in the search for DMD therapeutics. Several exon-skipping drugs employing RNA manipulation technology have been approved by regulatory agencies and have shown promise in clinical trials. This review summarizes recent scientific and clinical progress of ASO and other novel RNA manipulations, including RNA-based editing using MS2 coat protein-conjugated adenosine deaminase acting on the RNA (MCP-ADAR) system illustrating the efficacy and limitations of therapies to restore dystrophin. Perhaps lessons from this review will encourage the application of RNA-editing therapy to other neuromuscular disorders.

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Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is genetically altered in several cancers, including NSCLC, melanoma, lymphoma, and other tumors. Although ALK is associated with various cancers, the relationship between ALK expression and patient prognosis in different cancers is poorly understood. Here, using multidimensional approaches, we revealed the correlation between ALK expression and the clinical outcomes of patients with LUAD, melanoma, OV, DLBC, AML, and BC. We analyzed ALK transcriptional expression, patient survival rate, genetic alteration, protein network, and gene and microRNA (miRNA) co-expression. Compared to that in normal tissues, higher ALK expression was found in LUAD, melanoma, and OV, which are associated with poor patient survival rates. In contrast, lower transcriptional expression was found to decrease the survival rate of patients with DLBC, AML, and BC. A total of 202 missense mutations, 17 truncating mutations, 7 fusions, and 3 in-frame mutations were identified. Further, 17 genes and 19 miRNAs were found to be exclusively co-expressed and echinoderm microtubule-associated protein-like 4 (EML4) was identified as the most positively correlated gene (log odds ratio >3). The gene ontology and signaling pathways of the genes co-expressed with ALK in these six cancers were also identified. Our findings offer a basis for ALK as a prognostic biomarker and therapeutic target in cancers, which will potentially contribute to precision oncology and assist clinicians in identifying suitable treatment options.

Following the sequencing of the human genome, SNP analysis of individual patients has become essential for achieving the best drug response and ensuring optimal care. In this study, we developed a cost-effective probes-on-carrier DNA chip for the detection of SNPs. Our chips harbored three different probes against the TP53 gene, and were capable of detecting wild-type TP53 and SNPs such as rs121912651 and rs11540652. Four cell lines were used to validate the specificity of probe hybridization. Strong fluorescence intensity was observed in hybridized spots based on hybridization for perfect base pairing between complementary strands, whereas significantly lower fluorescence (p < 0.05) was observed in nonhybridized spots. These hybridization results indicate that the probes-on-carrier chip is suitable for SNP genotyping.