Precision oncology programs can rapidly identify oncogenic gene fusions in individual patients. However, despite their established oncogenic status, most gene fusions remain ‘undruggable’ due to the lack of specific inhibitory molecules. Here, I will present PspCas13b, a poorly characterized programmable RNA nuclease, which we established as a versatile tool to silence various oncogenic fusion transcripts1-2.
We first developed Single-Base Tiled crRNA screens (SiBTil), unbiased computational analysis, and comprehensive spacer-target mutagenesis approaches using >400 individual crRNAs, which uncovered the molecular basis of PspCas13 with single-base resolution. These approaches revealed that PspCas13b lacks any protospacer flanking sequences (PFS) that restrict the catalytic activity of other CRISPR enzymes. Interestingly, our data revealed that spacer nucleotide positions are key determinants of PspCas13b activity. Our de novo design of crRNAs harboring base-paired or mismatched guanosine bases at key spacer positions greatly enhances the silencing efficacy of otherwise inefficient crRNAs, expanding the targeting spectrum of this RNA silencing enzyme. We also reveal the interface between mismatch tolerance and intolerance of PspCas13b, which unlocked an unexpected single-base precision silencing capability of this RNA nuclease.
We demonstrate that 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. Collectively, this study provides new design principles for PspCas13b programming to specifically recognize and degrade any ‘undruggable’ fusion oncogenic transcript, thus providing a new conceptual framework for personalized oncology and beyond.
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