Oligo-CALL: A Next-Generation Barcoding Platform for Studying Resistance to Targeted Therapy [bulk RNA-seq]

Deconvolving heterogeneous cell populations and tracing individual clonal trajectories are essential for unraveling complex biological processes, such as therapy resistance. Although CRISPR-assisted cellular barcoding holds promises for lineage tracing, current platforms exhibit limitations underscored by suboptimal efficiencies and incompatibilities with single-cell transcriptomics. We introduce Oligo-CALL (Oligonucleotide-induced CRISPRa-Assisted Lineage Labeling), a new cellular barcoding system enabling precise tumor clone tracking, convenient live-clone isolation without additional genomic modification, and compatibility with single-cell RNA sequencing. Applying Oligo-CALL to human lung cancer cells revealed key insights into the resistance to KRAS G12C inhibitors (G12Ci). Clonal fate assays demonstrated that certain clones were repeatedly enriched under G12Ci pressure, indicating a “predestined” rather than “stochastic” mode of resistance. Paired functional assays of treatment-naïve versus resistant clones revealed clone-specific, acquired resistance phenotypes, manifesting as transient or permanent. Single-cell transcriptomics confirmed the activation of diverse yet clone-specific adaptive pathways underlying G12Ci resistance. Together, Oligo-CALL offers a powerful way to investigate the evolution of resistance to targeted therapies.