A Multimodal Arrayed CRISPRi Framework for Functional Characterization of lncRNAs Across Cellular Contexts

Background: Long non-coding RNAs (lncRNAs) are numerous, often lowly expressed, and highly context-dependent, making their functional characterization challenging using single-readout approaches. Integrating target repression assessment with complementary molecular and cellular phenotyping is therefore important for systematic lncRNA functional characterization across cellular contexts. Methods: To address this, we developed an arrayed CRISPR interference (CRISPRi)-based multimodal framework for functional lncRNA characterization across cellular contexts, pairing targeted knockdown with target repression assessment, shallow 3′-end RNA-seq, live-cell growth analysis, and Cell Painting-based morphological profiling. As a proof of concept, we applied this strategy to HEK293T and KELLY cells, targeting 68 expressed lncRNAs. Results: Targeted 3′-end RNA capture-seq stratified targets by baseline coverage and confirmed measurable repression for 18 lncRNAs in HEK293T cells and 21 lncRNAs in KELLY cells. Transcriptomic profiling captured the broadest layer of downstream response to lncRNA repression, whereas growth and morphology phenotypes were less frequent and restricted to a small subset of targets. Matched comparisons between HEK293T and KELLY showed that both molecular and cellular responses to the same lncRNA perturbation were often cell-line-dependent. As a representative multimodal hit, EPB41L4A-AS1 repression in KELLY cells was associated with reduced translation- and biosynthesis-related pathway activity, reduced cell growth, and morphological feature changes involving Golgi, actin and mitochondria. Conclusions: Together, these results demonstrate that arrayed CRISPRi coupled with multimodal profiling provides a structured framework for systematically characterizing lncRNA perturbation effects across complementary molecular and cellular readouts and prioritizing candidate functional lncRNAs for downstream mechanistic studies.