The type III-E CRISPR-controlled protease Craspase is distinguished from other type III systems by its single-subunit RNA-guided protein complex and direct coupling of RNA recognition to protease activation without second messenger signaling, making it an attractive development platform for bioengineering and therapeutics. Here, we identify five positions within the CRISPR RNA (crRNA) of Craspase from Candidatus "Scalindua brodae" (Sb-Craspase) that are sensitive to single-nucleotide mismatches. We leverage these positions to design crRNAs that selectively target clinically relevant single-nucleotide variants (SNVs) in oncogenic RNA transcripts. Using this approach, Sb-Craspase is selectively activated by the "undruggable" KRAS G12D SNV, while the wild-type transcript does not induce protease activation. Collectively, our results establish a framework for designing crRNAs to target clinically relevant SNVs, laying the groundwork for Craspase-based diagnostics and therapeutics against otherwise intractable oncogenic mutations. ... Read more