Cold Spring Harbor Laboratory

University of California

Merck + Co., Inc.

Merck Sharp + Dohme Corp.

The University of North Carolina at Chapel Hill

Cold Spring Harbor

The University of North Carolina

Gregory J Hannon

David H Beach

Manuel Serrano

Douglas J Demetrick

Sihem Cheloufi

Patrick Paddison

Scott Hammond

Douglas S Conklin

Amy Caudy

Emily Bernstein

Scott William Lowe

Lin He

Michele A Cleary

Stephen H Friend

Xingyue He

Ernest M Coffey

Ross Dickins

Peter S Linsley

Partha Pratim Mitra

Lee Lim

Charles J Sherr

Julja Burchard

Dawn E Quelle

Aimee L Jackson

Johannes Zuber

Jill F Magnus

Christof Fellmann

Douglas Conklin

J Michael Thomson

Peiqing Sun

Yong Jin Choi

Chao-Po Lin

Summer Goodson

John Michael Thomson

Kristopher A Kilian

Yaniv Erlich

Kristopher A Killian

Patrick J Paddison

Hammond Scott

Provided is an improved design of shRNA based on structural mimics of miR-451 precursors. These miR-451 shRNA mimics are channeled through a novel small RNA biogenesis pathway, require AGO2 catalysis and are processed by Drosha but are independent of DICER processing. This miRNA pathway feeds active elements only into Ago2 because of its unique catalytic activity. These data demonstrate that this newly identified small RNA biogenesis pathway can be exploited in vivo to produce active molecules.

Structurally designed shRNAs

Provided is an improved design of shRNA based on structural mimics of miR-451 precursors. These miR-451 shRNA mimics are channeled through a novel small RNA biogenesis pathway, require AGO2 catalysis and are processed by Drosha but are independent of DICER processing. This miRNA pathway feeds active elements only into Agog because of its unique catalytic activity. These data demonstrate that this newly identified small RNA biogenesis pathway can be exploited in vivo to produce active molecules.

The present disclosure provides a method of generating an induced pluripotent stem cell; as well as nucleic acids and genetically modified host cells useful in generating iPSCs. The present disclosure provides iPSCs, and methods of use of same.

Compositions and methods for generating induced pluripotent stem cells

Provided is a single construct combining a sequence encoding an RNAi molecule, a sequence encoding a reporter, and a target sequence specific for the RNAi molecule. The construct can be used to determine the potency of the encoded RNAi molecule in a direct and unbiased way. These results can be used to inform the design of potent RNAi molecules of various types and can be extended to several other applications, including: (1) generation of tiled libraries comprising every possible RNAi molecule-encoding sequence for a given gene target; (2) large-scale parallel validation of RNAi molecules targeting many genes to generate validated RNAi molecule-encoding libraries; (3) experimental comparison of design algorithms and strategies; and (4) investigation of RNAi biology in target site mutagenesis assays by screening pools containing single nucleotide changes in target sites and/or in the RNAi molecule to identify the most relevant sequence characteristics of potent RNAi-target site predictions.

High throughput methods for functionally determining RNA interference efficiency

The present invention provides methods for attenuating gene expression in a cell, especially in a mammalian cell, using gene-targeted double stranded RNA (dsRNA), such as a hairpin RNA. The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the 'target' gene).

Methods and compositions for RNA interference

The present invention provides methods of making a population of nucleic acid molecules, wherein each nucleic acid molecule comprises a predetermined nucleic acid sequence, each of said methods comprising the steps of: (a) synthesizing, on a substrate, a population of nucleic acid molecules wherein: i) each synthesized nucleic acid molecule comprises a predetermined nucleic acid sequence; and ii) each synthesized nucleic acid molecule is localized to a defined area of said substrate; (b) harvesting said population of synthesized nucleic acid molecules from said substrate to yield harvested nucleic acid molecules; and (c) introducing said harvested nucleic acid molecules into vector molecules.

Method for making populations of defined nucleic acid molecules

The invention relates to recombinant vectors for inducible and/or tissue specific expression of double-stranded RNA molecules that interfere with the expression of a target gene. In certain embodiments, the invention relates to the use of Tet (tetracycline)-responsive RNA Polymerase II (Pol II) promoters (e.g., TetON or TetOFF) to direct inducible knockdown in certain cells of an integrated or an endogenous gene, such as p53. The invention also relates to a method for producing transgenic animals (e.g., mice) expressing inducible (such as tetracycline-regulated), reversible, and/or tissue-specific double-stranded RNA molecules that interfere with the expression of a target gene.

Methods for producing microRNAs

Methods and systems of DNA sequencing that compensate for sources of noise in next-generation DNA sequencers are described.

Methods and systems of DNA sequencing

In one aspect, the invention generally relates to use of miR-34 as a biomarker to estimate TP53 function in a cell. In another aspect, the invention generally relates to multiple uses of miR-34 and siRNAs functionally and structurally related to miR-34 for the treatment of cancer.

Methods of using MIR34 as a biomarker for TP53 functional status

The present invention provides methods for attenuating gene expression in a cell using gene-targeted double stranded RNA (dsRNA). The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the 'target' gene).

Growing community of inventors

Huntington, NY, United States of America

Gregory J Hannon