Massachusetts Institute of Technology

University of Massachusetts

Max-planck-gesellschaft Zur F&#xf6;rderung Der Wissenschaften E. V.

Whitehead Institute for Biomedical Research

Harvard College

The Broad Institute, Inc.

Other

Whitehead Institute

Whitehead Insti.

Phillip A Sharp

Thomas Tuschl

Phillip D Zamore

David P Bartel

Carl O Pabo

Joel L Pomerantz

Michael J Lenardo

Albert S Baldwin

David Baltimore

Thomas P Maniatis

Roger G Clerc

Jonathan H Lebowitz

Louis Staudt

Harinder Singh

Ranjan Sen

Lynn M Corcoran

Patrick A Baeuerle

Chen-Ming Fan

Feng Zhang

Sidi Chen

Paul Chang

Anthony Leung

Jeremy E Wilusz

Neville Espi Sanjana

Constance L Cepko

Mario R Capecchi

Randall Jeffrey Platt

Qiang Zhou

Uttam L Rajbhandary

Sejal K Vyas

Dig Mahat

Paul Changelian

Frank A Laski

Philip D Zamore

The present invention relates to in vivo methods for modeling tumor formation and/or tumor evolution comprising the use of eukaryotic cells in which one or more genetic target locus has been altered by the CRISPR/Cas system, and which cells are transplanted in non-human eukaryote as a model system for tumor formation and tumor evolution. In particular in vivo genetic screening methods for identifying genes involved in tumorigenesis and metastasis are disclosed. The invention further relates to kits and components for practicing the methods, as well as materials obtainable by the methods, in particular tumor and metastasis samples and cells or cell lines derived therefrom. The invention also relates to diagnostic and therapeutic methods derived from the information obtained in the modeling methods.

CRISPR mediated in vivo modeling and genetic screening of tumor growth and metastasis

The present disclosure relates to a method of sequencing nascent RNA in a cell. In some embodiments, the nascent RNA is conjugated to DNA using copper-catalyzed azide-alkyne cycloaddition (CuAAC). Methods of the present disclosure can be used to generate genomic libraries of a cell and measure gene expression and enhancer and/or super-enhancer activity.

Single-cell RNA sequencing using click-chemistry

The invention involves inducing 3-50 or more mutations (e.g., any whole number between 3 and 50 of mutations, with it noted that in some embodiments there can be up to 16 different RNA(s), e.g., sgRNAs each having its own a promoter, in a vector, such as AAV, and that when each sgRNA does not have its own promoter, there can be twice to thrice that amount of different RNA(s), e.g., sgRNAs, e.g., 32 or even 48 different guides delivered by one vector) in transgenic Cas9 eukaryotes to model genetic disease, e.g. cancer. The invention comprehends testing putative treatments with such models, e.g., testing putative chemical compounds that may be pharmaceutically relevant for treatment or gene therapy that may be relevant for treatment, or combinations thereof. The invention allows for the study of genetic diseases and putative treatments to better understand and alleviate a genetic disease or a condition, e.g., cancer.

Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for modeling competition of multiple cancer mutations in vivo

The invention relates in aspects to hybrid RNAs lacking a poly-A tail and nucleic acid vectors for expressing the RNA. The hybrid RNAs in some instances have a stabilizing triple helical structure. Related methods for expressing RNA in vivo and in vitro are also disclosed.

Production of stable non-polyadenylated RNAs

The present invention relates to ain vitro system which was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length. Furthermore, when these 21-23 nt fragments are purified and added back toextracts, they mediate RNA interference in the absence of long dsRNA. Thus, these 21-23 nt fragments are the sequence-specific mediators of RNA degradation. A molecular signal, which may be their specific length, must be present in these 21-23 nt fragments to recruit cellular factors involved in RNAi. This present invention encompasses these 21-23 nt fragments and their use for specifically inactivating gene function. The use of these fragments (or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for degradation in mammalian cells, where the use of long dsRNAs to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response. This specific targeting of a particular gene function is useful in functional genomic and therapeutic applications.

RNA sequence-specific mediators of RNA interference

The invention relates in aspects to hybrid RNAs lacking a poly-A tail and nucleic acid vectors for expressing the RNA. The hybrid RNAs in some instances have a 3' terminal stabilizing triple helical structure. Related methods for expressing said RNAs in vivo and in vitro are also disclosed.

The invention provides methods for treating or decreasing the likelihood of developing a stress-granule related disorder and/or cancer by administering one or more poly-ADP-ribose polymerase (PARP) inhibitors, one or more PARP activators, one or more poly-ADP-ribose glycosylase (PARG) activators, and/or one or more poly-ADP-ribose glycohydrolase ARH3 activators. The invention also provides corresponding methods of decreasing stress granule formation and/or proliferation in a cell or a population of cells. The invention further provides methods of increasing the number of stress granules and proliferation in a cell or a population of cells by administering one or more PARP activators, one or more PARP inhibitors, one or more PARG inhibitors, and/or one or more ARH3 inhibitors. The invention also provides methods for screening for agents for treating or decreasing the likelihood of developing a stress granule-related disorder or cancer, and methods for determining the propensity for developing a stress granule-related disorder or cancer, as well as compositions and kits containing one or more PARP inhibitors, one or more PARP activators, one or more PARG activators, and one or more ARH3 activators.

Compositions and methods for treating cancer and modulating stress granule formation

The present invention relates to a Drosophila in vitro system which was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length. Furthermore, when these 21-23 nt fragments are purified and added back to Drosophila extracts, they mediate RNA interference in the absence of long dsRNA. Thus, these 21-23 nt fragments are the sequence-specific mediators of RNA degradation. A molecular signal, which may be their specific length, must be present in these 21-23 nt fragments to recruit cellular factors involved in RNAi. This present invention encompasses these 21-23 nt fragments and their use for specifically inactivating gene function. The use of these fragments (or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for degradation in mammalian cells, where the use of long dsRNAs to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response. This specific targeting of a particular gene function is useful in functional genomic and therapeutic applications.

The invention provides methods for increasing the activity of an inhibitory RNA (RNAi) in a subject requiring administering one or more poly-ADP-ribose polymerase (PARP) inhibitors and/or one or more PARG activators to the subject. The invention also provides methods for increasing the activity of an inhibitory RNA in a cell or cell population requiring contacting a cell or cell population with one or more PARP inhibitors and/or one or more PARG activators. The invention further provides compositions and kits containing one or more PARP inhibitors and/or one or more PARG activators.

Methods and compositions for increasing the activity of inhibitory RNA

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Newton, MA, United States of America

Phillip A Sharp