Abstract: A method of nucleic acid analysis is described, the method including the steps of (a) providing a sample comprising a plurality of end-blocked polynucleotides derived from a biological source; (b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that targets a sequence of interest to produce polynucleotide fragments that comprise the sequence of interest and a ligatable end generated by endonuclease cleavage; (c) ligating a moiety to the ligatable end to produce a moiety-target polynucleotide construct; and (d) detecting the moiety-target polynucleotide construct or a transcription or translation produce produced from the moiety-target polynucleotide construct using mass spectrometry. The moiety may be an adaptor sequence with a promoter for RNA polymerase. The moiety may be a chemical moiety that is highly amenable to flight and detection in a mass spectrometer.

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.

Abstract: A method of sample analysis is described comprising providing a sample comprising a plurality of endblocked polynucleotides, digesting the sample with one or more defined nucleic acid-directed endonuclease that targets a sequence of interest to produce a digested sample of polynucleotide fragments, wherein one or more of the fragments in the digested sample comprises: a sequence of interest and at least one ligatable end that has been generated by endonuclease cleavage; (c) enriching for fragments that contain the sequence of interest, wherein the one or more sequences of interest are enriched greater than 55 times, or greater than 750 times, their relative abundance in the sample; and (d) analyzing the enriched sample.

Abstract: A method of sample analysis is provided. In some embodiments, the method comprises: (a) digesting a mixed nucleic acid sample with a plurality of reprogrammed nucleic acid-directed endonucleases that target sequences of interest to produce a digested sample, wherein at least some of the fragments in the digested sample comprise: (i) a sequence of interest and (ii) at least one ligatable end that has been generated by endonuclease cleavage; (b) enriching for fragments that contain the sequence of interest; and (c) analyzing the enriched fragments. Kits for performing the method are also provided.

Abstract: A method of sample analysis is provided. In some embodiments, the method comprises: (a) digesting a mixed nucleic acid sample with a plurality of reprogrammed nucleic acid-directed endonucleases that target sequences of interest to produce a digested sample, wherein at least some of the fragments in the digested sample comprise: (i) a sequence of interest and (ii) at least one ligatable end that has been generated by endonuclease cleavage; (b) enriching for fragments that contain the sequence of interest; and (c) analyzing the enriched fragments. Kits for performing the method are also provided.

Abstract: Among other things, this disclosure describes a method comprising: cleaving a plurality of target sequences in an adaptor-tagged sequencing library using population of reprogrammed nucleic acid-directed endonucleases; non-specifically amplifying the library, thereby amplifying fragments that have not been cleaved; and sequencing the amplified sample.