Molecular principles of CRISPR-Cas13b design for selective silencing of oncogenic fusion transcripts
Oncogenic gene fusions are key drivers of cancer, yet most remain untargetable by current therapies. Here, we establish CRISPR-PspCas13b as a personalizable platform for systematic silencing of diverse fusion transcripts. We show that recognition and cleavage of the breakpoint sequence by PspCas13b disrupts the fusion transcript, resulting in efficient protein depletion. Short- and long-read RNA sequencing reveal that PspCas13b cleavage generates aberrant, translation-incompetent RNA species with preserved 5' and 3' ends, and internal deletions proximal to the cleavage site. Importantly, targeting the BCR::ABL1 breakpoint sequence efficiently degrades both canonical and drug-resistant BCR::ABL1 mutants, which are the primary drivers of tyrosine kinase inhibitor (TKI) resistance and relapse in chronic myeloid leukemia (CML). Molecular profiling shows that PspCas13b impairs BCR::ABL1 tyrosine phosphokinase activity and triggers extensive transcriptomic and proteomic remodeling, ultimately leading to erythroid differentiation and apoptosis in CML cells fully resistant to clinical drugs. Beyond BCR::ABL1 mutants, personalized design of PspCas13b effectively silences other undruggable fusions, including RUNX1::RUNX1T1 and EWSR1::FLI1, which drive acute myeloid leukemia and Ewing's sarcoma, respectively. Collectively, this study establishes a molecular framework for systematic, precise, and personalizable targeting of otherwise undruggable or drug-resistant oncogenic transcripts.