Biology subjects

Casan, J. M.

Publications and source records attributed to Casan, J. M..

3 recordsLinked to original sources

bioRxiv2025

Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

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 various fusion transcripts. We reveal that recognition and cleavage of the breakpoint sequence by PspCas13b disrupts the fusion transcript, resulting in unexpected RNA nicking and ligation near the cleavage site, which generates out-of-frame, translation-incompetent transcripts. This approach efficiently degrades canonical and drug-resistant BCR::ABL1 mutants (e.g., T315I), a primary cause of resistance to tyrosine kinase inhibitors (TKIs) and relapse in chronic myeloid leukemia (CML). Silencing T315I BCR::ABL1 mRNA in drug-resistant CML cells triggers extensive transcriptomic, proteomic, and phosphoproteomic remodelling, causing erythroid differentiation and apoptosis. Beyond BCR-ABL1 mutants, personalized design of PspCas13b effectively silences other undruggable fusions, including RUNX1::RUNX1T1 and EWSR1::FLI1, key drivers in acute myeloid leukemia and in Ewing sarcoma, respectively. Collectively, this study establishes a framework for systematic, precise, and personalizable targeting of otherwise undruggable or drug-resistant oncogenic transcripts.

molecular biology↗

bioRxiv2022

Single-base precision design of CRISPR-Cas13b enables systematic silencing of oncogenic fusions

Precision oncology programs can rapidly identify oncogenic gene fusions in individual patients1-3. However, despite their established oncogenic status, the vast majority of gene fusions remain undruggable due to the lack of specific inhibitory molecules4, 5. Here, we establish PspCas13b, a poorly characterized programmable RNA nuclease, as a versatile tool to silence various oncogenic fusion transcripts. Our Single-Base Tiled crRNA screens (SiBTil), unbiased computational analysis, and comprehensive spacer-target mutagenesis revealed key determinants of PspCas13b activity. De novo design of crRNAs harbouring basepaired or mismatched guanosine bases at key spacer positions greatly enhances the silencing efficacy of otherwise inefficient crRNAs, expanding the targeting spectrum of this enzyme. We also reveal the interface between mismatch tolerance and intolerance, which unlocks an unexpected single-base precision targeting capability of this RNA nuclease. Notably, our de novo design principles enable potent and selective silencing of various gene fusion transcripts and their downstream oncogenic networks, without off-targeting of non-translocated variants that share extensive sequence homology. We demonstrate that PspCas13b targeting the breakpoint of fusion transcripts enables efficient suppression of ancestral and single-nucleotide mutants (e.g. BCR-ABL1 T315I) that often drive clinical cancer relapse. Collectively, this study provides new design principles for PspCas13b programming to specifically recognise and degrade any undruggable fusion oncogenic transcript, thus providing a new conceptual framework for personalized oncology.

molecular biology↗

bioRxiv2020

Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance

Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.

microbiology↗