University of California

Mitchell R O'Connell

Jennifer A Doudna

James Harrison Doudna Cate

Spencer Charles Knight

Alexandra East-Seletsky

Samuel H Sternberg

Benjamin L Oakes

The present disclosure provides methods for detecting a single-stranded target RNA. The present disclosure provides methods of cleaving a precursor C2c2 guide RNA array into two or more C2c2 guide RNAs. The present disclosure provides a kit for detecting a target RNA in a sample.

Methods and compositions for detecting a target RNA

The present disclosure provides compositions and methods for labeling a single stranded target nucleic acid. Subject compositions include a Cas9 protein, a Cas9 guide RNA, and a quenched PAMmer. A subject quenched PAMmer is a single stranded oligonucleotide having (i) a protospacer adjacent motif (PAM) sequence; (ii) a detectable label; (iii) a quencher moiety that quenches the detectable label; and (iv) at least one of: a specificity segment positioned 5' of the PAM sequence, and an orientation segment positioned 3&#x2032; of the PAM sequence. In the subject methods, the Cas9 protein cleaves the quenched PAMmer at a cleavage site positioned between the detectable label and the quencher moiety to produce: (a) a first cleavage product that is hybridized with the target nucleic acid and comprises the detectable label; and (b) a second cleavage product that is not hybridized with the target nucleic acid and comprises the quencher moiety.

Methods and compositions for labeling a single-stranded target nucleic acid

The present disclosure provides compositions and methods for binding and/or cleaving a single stranded target nucleic acid. Subject compositions include a Cas9 polypeptide, a guide nucleic acid, and a PAMmer. A subject PAMmer is a single stranded oligonucleotide having a protospacer adjacent motif (PAM) sequence and at least one of: a specifity segment positioned 5' of the PAM sequence, and an orientation segment positioned 3&#x2032; of the PAM sequence. In some embodiments, the Cas9 polypeptide is a variant Cas9 polypeptide having reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide. In some cases, methods of binding are for visualizing single stranded target nucleic acids using a detectable label. In some cases, methods of binding are for isolating, collecting, and/or analyzing at least one of: (i) bound single stranded target nucleic acids; and (ii) polypeptides associated with bound single stranded target nucleic acids.

Growing community of inventors

Berkeley, CA, United States of America

Mitchell R O'Connell