CRISPR-Cas12a (Cpf1) proteins, part of the bacterial adaptive immune system, are RNA-guided DNA targeting enzymes that can induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. This study reveals that RNA-guided DNA binding in Cas12a triggers robust, non-specific single-stranded DNA (ssDNA) cleavage activity, sufficient to completely degrade both linear and circular ssDNA molecules. This activity, catalyzed by the same active site responsible for dsDNA cutting, is a fundamental property of other type V CRISPR-Cas12 enzymes. Activation of ssDNA cutting requires precise recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence. The researchers combined this target-dependent Cas12a ssDNase activation with isothermal amplification to create a method called DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), achieving attomolar sensitivity for nucleic acid detection. DETECTR enables rapid and specific detection of HPV in human patient samples, providing a simple platform for nucleic acid-based, point-of-care diagnostics. Cas12a and related type V CRISPR interference proteins unleash non-specific, single-stranded DNase activity upon guide RNA-dependent DNA binding, which can be harnessed for rapid and specific nucleic acid detection. The study also shows that Cas12a can cleave ssDNA regardless of complementarity to the guide RNA, and that the PAM sequence is critical for catalytic activation by a crRNA-complementary dsDNA but not for a crRNA-complementary ssDNA. The findings suggest that target-dependent activation of non-specific ssDNA cleavage is a fundamental feature of all type V CRISPR-Cas12 proteins. DETECTR was tested for HPV detection and demonstrated high sensitivity and specificity, accurately identifying HPV types 16 and 18 in patient samples. The study highlights the potential of Cas12a for improving the speed, sensitivity, and specificity of nucleic acid detection in point-of-care diagnostic applications.CRISPR-Cas12a (Cpf1) proteins, part of the bacterial adaptive immune system, are RNA-guided DNA targeting enzymes that can induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. This study reveals that RNA-guided DNA binding in Cas12a triggers robust, non-specific single-stranded DNA (ssDNA) cleavage activity, sufficient to completely degrade both linear and circular ssDNA molecules. This activity, catalyzed by the same active site responsible for dsDNA cutting, is a fundamental property of other type V CRISPR-Cas12 enzymes. Activation of ssDNA cutting requires precise recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence. The researchers combined this target-dependent Cas12a ssDNase activation with isothermal amplification to create a method called DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), achieving attomolar sensitivity for nucleic acid detection. DETECTR enables rapid and specific detection of HPV in human patient samples, providing a simple platform for nucleic acid-based, point-of-care diagnostics. Cas12a and related type V CRISPR interference proteins unleash non-specific, single-stranded DNase activity upon guide RNA-dependent DNA binding, which can be harnessed for rapid and specific nucleic acid detection. The study also shows that Cas12a can cleave ssDNA regardless of complementarity to the guide RNA, and that the PAM sequence is critical for catalytic activation by a crRNA-complementary dsDNA but not for a crRNA-complementary ssDNA. The findings suggest that target-dependent activation of non-specific ssDNA cleavage is a fundamental feature of all type V CRISPR-Cas12 proteins. DETECTR was tested for HPV detection and demonstrated high sensitivity and specificity, accurately identifying HPV types 16 and 18 in patient samples. The study highlights the potential of Cas12a for improving the speed, sensitivity, and specificity of nucleic acid detection in point-of-care diagnostic applications.