Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions between Non-Coding RNAs in Cancer Cells

Abstract

Long non-coding RNAs (lncRNAs) represent a vast and functionally diverse class of RNA molecules, with over 100,000 predicted in the human genome. Although lncRNAs are less conserved across species compared to protein-coding genes, they play critical roles in gene regulation, chromatin interactions, and cancer progression. Their involvement in cancer make them promising therapeutic targets. CRISPR interference (CRISPRi), utilizing catalytically inactive Cas9 fused with a transcriptional repressor such as KRAB-MeCP2, offers a precise method for targeting nuclear lncRNAs and assessing their functions. This study introduces a dual CRISPRi system using orthogonal CRISPRi technologies from Staphylococcus aureus and Streptococcus pyogenes dCas9-KRAB, optimized for combinatorial targeting of lncRNAs in human melanoma cells. The protocol facilitates combinatorial gene knockdown or synthetic lethal screening of lncRNA pairs, providing a novel tool for cancer research. By exploring synthetic lethality between lncRNAs, this approach can help identify lncRNA interactions critical for cancer cell survival, offering new therapeutic strategies. The dual systems functionality is demonstrated, highlighting its potential in identifying critical cancer-specific lncRNA interactions. SUMMARYThis study presents a dual CRISPRi system targeting long non-coding RNAs in melanoma cells. It enables combinatorial gene knockdown and synthetic lethal screening, identifying cancerspecific lncRNA interactions for potential therapeutic strategies.