Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition.

Oncogenic gene fusions are key drivers of cancer, yet most remain undruggable by current therapies. Here, we establish CRISPR-PspCas13b as a personalizable platform for systematic silencing of various fusion transcripts. We reveal that recognition and cleavage of the breakpoint sequence by PspCas13b disrupt the fusion transcript, resulting in efficient protein depletion. PspCas13b cleavage produces aberrant, translation-incompetent target RNA species, a subset of which harbors internal deletions proximal to the cleavage site. We demonstrate that targeting the BCR::ABL1 breakpoint sequence efficiently degrades both wild-type and drug-resistant BCR::ABL1 mutants, which are the primary drivers of tyrosine kinase inhibitor resistance and relapse in chronic myeloid leukemia. Molecular profiling revealed that PspCas13b impairs BCR::ABL1 tyrosine phosphokinase activity and triggers extensive transcriptomic and proteomic remodeling, leading to erythroid differentiation and apoptosis in drug-resistant cells. Beyond BCR::ABL1, personalized design of PspCas13b effectively silences other undruggable oncogenic fusions. Collectively, this study establishes a molecular framework for precise targeting of undruggable or drug-resistant oncogenic transcripts.