University of California

Mammoth Biosciences, Inc.

Lucas Benjamin Harrington

Jennifer A Doudna

Jillian F Banfield

David Burstein

Janice S Chen

David Paez-Espino

Enbo Ma

Steven C Strutt

James Paul Broughton

Wiputra Jaya Hartono

Janice Sha Chen

Jasmeet Singh

Pedro Patrick Draper Galarza

Xin Miao

Andrew Besancon Lane

William Douglass Wright

Aaron DeLoughery

Maria-Nefeli Tsaloglou

Clarissa Oriel Rhines

Clare Louise Fasching

Benjamin Julius Rauch

Ashley Tehranchi

Nikos C Kyrpides

Matan Drory Retwitzer

Alexander Richard Neckelmann

Stepan Tymoshenko

Fnu Yunanda

Isaac Paterson Witte

Provided are compositions and methods for detecting a target DNA (double stranded or single stranded) in a sample. In some embodiments, a subject method includes: (a) contacting the sample with: (i) a type V CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e); (ii) a guide RNA (comprising a region that binds to the type V CRISPR/Cas effector protein, and a guide sequence that hybridizes with the target DNA); and (iii) a detector DNA that is single stranded (i.e., a 'single stranded detector DNA') and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal produced by cleavage (by the type V CRISPR/Cas effector protein) of the single stranded detector DNA. Also provided are compositions and methods for cleaving single stranded DNAs (e.g., non-target ssDNAs), e.g., inside of a cell.

Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs

Provided are compositions and methods that include one or more of: (1) a 'CasZ' protein (also referred to as a CasZ polypeptide), a nucleic acid encoding the CasZ protein, and/or a modified host cell comprising the CasZ protein (and/or a nucleic acid encoding the same); (2) a CasZ guide RNA that binds to and provides sequence specificity to the CasZ protein, a nucleic acid encoding the CasZ guide RNA, and/or a modified host cell comprising the CasZ guide RNA (and/or a nucleic acid encoding the same); and (3) a CasZ transactivating noncoding RNA (trancRNA) (referred to herein as a “CasZ trancRNA”), a nucleic acid encoding the CasZ trancRNA, and/or a modified host cell comprising the CasZ trancRNA (and/or a nucleic acid encoding the same).

CasZ compositions and methods of use

The present disclosure provides CasY proteins, nucleic acids encoding the CasY proteins, and modified host cells comprising the CasY proteins and/or nucleic acids encoding same. CasY proteins are useful in a variety of applications, which are provided. The present disclosure provides CasY guide RNAs that bind to and provide sequence specificity to the CasY proteins, nucleic acids encoding the CasY guide RNAs, and modified host cells comprising the CasY guide RNAs and/or nucleic acids encoding same. CasY guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides methods of identifying a CRISPR RNA-guided endonuclease.

RNA-guided nucleic acid modifying enzymes and methods of use thereof

Provided are compositions and methods that include a CasY transactivating noncoding RNA (trancRNA) (referred to herein as a 'CasY trancRNA'), nucleic acids encoding the CasY trancRNA, and/or a modified host cell comprising the CasY trancRNA (and/or a nucleic acid encoding the same). Subject compositions and methods can also include one or more of: (a) a “CasY” protein (also referred to as a CasY polypeptide, a Cas12d protein, and a Cas12d polypeptide), a nucleic acid encoding the CasY protein, and/or a modified host cell comprising the CasY protein (and/or a nucleic acid encoding the same); and (b) a CasY guide RNA (also referred to herein as a “Cas12d guide RNA”) that binds to and provides sequence specificity to the CasY protein, a nucleic acid encoding the CasY guide RNA, and/or a modified host cell comprising the CasY guide RNA (and/or a nucleic acid encoding the same).

CasY compositions and methods of use

Provided herein are compositions, systems, and methods comprising effector proteins and uses thereof. These effector proteins are shown to be active with guide RNAs and may be characterized as CRISPR-associated (Cas) proteins. Various compositions, systems, and methods of the present disclosure leverage the activities of these effector proteins for the modification, detection, and engineering of nucleic acids.

Effector proteins and methods of use

Provided are compositions and methods that include one or more of: (1) a Class 2 CRISPR/Cas effector protein, a nucleic acid encoding the effector protein, and/or a modified host cell comprising the effector protein (and/or a nucleic acid encoding the same); (2) a CRISPR/Cas guide RNA that binds to and provides sequence specificity to the Class 2 CRISPR/Cas effector protein, a nucleic acid encoding the CRISPR/Cas guide RNA, and/or a modified host cell comprising the CRISPR/Cas guide RNA (and/or a nucleic acid encoding the same); and (3) a CRISPR/Cas transactivating noncoding RNA (trancRNA), a nucleic acid encoding the CRISPR/Cas trancRNA, and/or a modified host cell comprising the CRISPR/Cas trancRNA (and/or a nucleic acid encoding the same).

Class 2 CRISPR/Cas compositions and methods of use

The present disclosure provides CasX proteins, nucleic acids encoding the CasX proteins, and modified host cells comprising the CasX proteins and/or nucleic acids encoding same. CasX proteins are useful in a variety of applications, which are provided. The present disclosure provides CasX guide RNAs that bind to and provide sequence specificity to the CasX proteins, nucleic acids encoding the CasX guide RNAs, and modified host cells comprising the CasX guide RNAs and/or nucleic acids encoding same. CasX guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides archaeal Cas9 polypeptides and nucleic acids encoding same, as well as their associated archaeal Cas9 guide RNAs and nucleic acids encoding same.

Described herein are devices, systems, fluidic devices, kits, and methods for detection of target nucleic acids associated with diseases, cancers, genetic disorders, a genotype, a phenotype, or ancestral origin. The devices, systems, fluidic devices, kits, and methods may comprise reagents of a guide nucleic acid targeting a target nucleic acid, a programmable nuclease, and a single stranded detector nucleic acid with a detection moiety. The target nucleic acid of interest may be indicative of a disease, and the disease may be communicable diseases, or of a cancer or genetic disorder. The target nucleic acid of interest may be indicative of a genotype, a phenotype, or ancestral origin.

Programmable nuclease compositions and methods of use thereof

The present disclosure provides RNA-guided endonucleases, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided endonuclease of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided endonuclease of the present disclosure and a guide RNA.

Thermostable RNA-guided endonucleases and methods of use thereof

Provided are compositions and methods that include one or more of: (1) a 'CasZ' protein (also referred to as a CasZ polypeptide), a nucleic acid encoding the CasZ protein, and/or a modified host cell comprising the CasZ protein (and/or a nucleic acid encoding the same); (2) a CasZ guide RNA that binds to and provides sequence specificity to the CasZ protein, a nucleic acid encoding the CasZ guide RNA, and/or a modified host cell comprising the CasZ guide RNA (and/or a nucleic acid encoding the same); and (3) a CasZ transactivating noncoding RNA (trancRNA) (referred to herein as a 'CasZ trancRNA'), a nucleic acid encoding the CasZ trancRNA, and/or a modified host cell comprising the CasZ trancRNA (and/or a nucleic acid encoding the same).

Growing community of inventors

Berkeley, CA, United States of America

Lucas Benjamin Harrington