University of California

Chen, Janice S.

Samuel H Sternberg

Jennifer A Doudna

Martin Jinek

Janice S Chen

Mitchell R O'Connell

Benjamin L Oakes

Emine Kaya

Fuguo Jiang

Addison V Wright

David W Taylor, Jr

Benjamin LaFrance

David Taylor

The present disclosure provides a Cas9 heterodimer, as well as nucleic acids encoding the Cas9 heterodimer, and host cells comprising the nucleic acids. The present disclosure provides a system that includes a Cas9 heterodimer of the present disclosure and at least one of: a Cas9 guide RNA, and a dimerizing agent. A Cas9 heterodimer of the present disclosure is useful in a wide variety of applications, which are also provided.

Heterodimeric Cas9 and methods of use thereof

The present disclosure provides variant Cas9 proteins, nucleic acids encoding the variant Cas9 proteins, and host cells comprising the nucleic acids. The present disclosure provides systems that include a subject variant Cas9 protein (and/or a nucleic acid encoding the variant Cas9 protein) and a Cas9 guide RNA. In some cases, a subject system includes a PAMmer and/or a donor polynucleotide. The variant Cas9 proteins and the nucleic acids encoding the variant Cas9 proteins are useful in a wide variety of methods, which are also provided. In some embodiments, a variant Cas9 protein includes a RuvC domain, an HNH domain, and a disrupted RuvC/HNH linker region that reduces the RuvC cleavage activity of the protein. In some embodiments, a variant Cas9 protein includes a deletion (of all or a part of the HNH domain) or an insertion (within the HNH domain) that reduces the HNH cleavage activity of the protein.

Cas9 variants and methods of use thereof

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.

Methods and compositions for modifying a single stranded target nucleic acid

The present disclosure provides atomic structures of Cas9 with and without polynucleotides bound thereto. Also provided is a computer-readable medium comprising atomic coordinates for Cas9 polypeptides in both an unbound configuration and a configuration wherein the Cas9 polypeptide is bound to one or more polynucleotides. The present disclosure provides crystals comprising Cas9 polypeptides; and compositions comprising the crystals. The present disclosure provides methods for the engineering of Cas9 polypeptides wherein Cas9 activity has been altered, ablated, or preserved and amended with additional activities.

Cas9 crystals and methods of use thereof

The present disclosure provides variant Cas endoribonucleases, nucleic acids encoding the variant Cas endoribonucleases, and host cells genetically modified with the nucleic acids. The variant Cas endoribonucleases find use in a variety of applications, which are also provided. The present disclosure also provides methods of detecting a specific sequence in a target polyribonucleotide; and methods of regulating production of a target RNA in a eukaryotic cell.

Growing community of inventors

El Cerrito, CA, United States of America

Samuel H Sternberg