METHODS AND COMPOSITIONS FOR MODIFYING A SINGLE STRANDED TARGET NUCLEIC ACID

摘要

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 specificity segment positioned 5′ of the PAM sequence, and an orientation segment positioned 3′ 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.

主权项

1. A method of cleaving a single stranded target nucleic acid, the method comprising:
contacting the single stranded target nucleic acid with:
(i) a Cas9 polypeptide;(ii) a guide nucleic acid comprising: (a) a targeting segment comprising a nucleotide sequence that is complementary to a first target nucleotide sequence in the single stranded target nucleic acid, and (b) a protein-binding segment comprising two stretches of nucleotides that hybridize with one another to form a double-stranded RNA duplex that interacts with the Cas9 polypeptide; and
(iii) a PAMmer, wherein the PAMmer is a single stranded oligonucleotide comprising:
(a) a protospacer adjacent motif (PAM) sequence, and(b) at least one of: (i) an orientation segment, positioned 3′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a second target nucleotide sequence in the target nucleic acid; and (ii) a specificity segment, positioned 5′ of the PAM sequence, comprising a nucleotide sequence that is complementary to said first target nucleotide sequence.