University of California

Benjamin L Oakes

Jennifer A Doudna

Mitchell R O'Connell

Samuel H Sternberg

David Frank Savage

Junjie Liu

Rayka Yokoo

Natalia Orlova

Dana Nadler

Abraham I Flamholz

The present disclosure provides variant type V CRISPR/Cas effector polypeptides, fusion polypeptides comprising the variant type V CRISPR/Cas effector polypeptides, and nucleic acids comprising nucleotide sequences encoding the variant polypeptides and fusion polypeptides. The present disclosure provides methods of binding, or binding and nicking, a target nucleic acid, using a variant type V CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides methods of detecting a single-stranded DNA, using a variant type V CRISPR/Cas effector polypeptide of the present disclosure.

Variant type V CRISPR/Cas effector polypeptides and methods of use thereof

The present disclosure provides (i) RNA-guided polypeptides (e.g., circular permuted Cas9 proteins) in which the N-terminal end of an N-terminal fragment of a parent RNA-guided polypeptide (e.g., a parent Cas9 protein) is fused (e.g., via linker) to the C-terminal end of the C-terminal fragment (thereby generating new N- and C-termini), (ii) conditionally active RNA-guided polypeptides (e.g., conditionally active circular permuted Cas9 proteins), and (iiii) Cas9 fusion polypeptides that include an internal insertion of a heterologous polypeptide; as well as methods that employ the above polypeptides.

Variant RNA-guided polypeptides and methods of use

The present disclosure provides variant Cas9 polypeptides, where a variant Cas9 polypeptide of the present disclosure comprises an internal insertion of a heterologous polypeptide. The present disclosure provides nucleic acids comprising nucleotide sequences encoding the variant Cas9 polypeptides. The present disclosure provides host cells comprising a variant Cas9 polypeptide of the present disclosure, or comprising a nucleic acid encoding a variant Cas9 polypeptide of the present disclosure. The present disclosure provides methods of binding and/or modifying a target nucleic acid, involving use of a variant Cas9 polypeptide of the present disclosure.

Variant Cas9 polypeptides comprising internal insertions

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

Benjamin L Oakes