Abstract: This disclosure provides methods and materials for determining whether a particular nucleic acid sequence is present in a test sample. Nucleic acid in the sample is treated with a nuclease mixture that generates monoribonucleotides if the target sequence is present. An exemplary nuclease mixture is a CRISPR associated protein that has endonuclease collateral activity (such as Cas12a), in combination with a guide RNA complementary to the target sequence, a reporter oligonucleotide, and an exonuclease. Upon binding of the Cas/gRNA complex to the target sequence in the sample, the endonuclease activity of the Cas protein cleaves the reporter oligonucleotide internally, rendering it suitable for digestion by the exonuclease. Monoribonucleotides that are generated as a consequence are measured by a bioluminescence detection means such as luciferase. The assay method is sufficiently robust, sensitive, and specific for use with a variety of biological test samples, sometimes without amplification.