Massachusetts Institute of Technology

Harvard College

The Broad Institute, Inc.

The University of Tokyo

Other

Inria (french Institute for Research in Computer Science and Automation)

Tsinghua University

Yinqing Li

Feng Zhang

Ron Weiss

Le Cong

Winston X Yan

Fei Ran

Osamu Nureki

Hiroshi Nishimasu

Aviv Regev

David A Weitz

Patrick D Hsu

David Arthur Scott

Xavier Duportet

Yun Long Jiang

Anindita Basu

Matthias Heidenreich

He Chen

Zhen Xie

Joshua Asher Weinstein

Lukasz Swiech

Naomi Habib

Gregory Batt

Weixi Liao

Kaijie Zheng

Inbal Avraham Davidi

Yun Jiang

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems. In particular the present invention comprehends engineered new guide architectures and enzymes to be used in optimizedCRISPR-Cas enzyme systems.

Engineering and optimization of systems, methods, enzymes and guide scaffolds of CAS9 orthologs and variants for sequence manipulation

Transcriptomes of individual neurons provide rich information about cell types and dynamic states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation from dense adult tissue is challenging, and markers for each phase are limited. Here, Applicants developed Nuc-seq, Div-Seq, and Dronc-Seq. Div-seq combines Nuc-Seq, a scalable single nucleus RNA-Seq method, with EdU-mediated labeling of proliferating cells. Nuc-Seq can sensitively identify closely related cell types within the adult hippocampus. Div-Seq can track transcriptional dynamics of newborn neurons in an adult neurogenic region in the hippocampus. Dronc-Seq uses a microfluidic device to co-encapsulate individual nuclei in reverse emulsion aqueous droplets in an oil medium together with one uniquely barcoded mRNA-capture bead. Finally, Applicants found rare adult newborn GABAergic neurons in the spinal cord, a non-canonical neurogenic region. Taken together, Nuc-Seq, Div-Seq and Dronc-Seq allow for unbiased analysis of any complex tissue.

Methods for determining spatial and temporal gene expression dynamics in single cells

Disclosed are thermodynamic and multiplication methods concerning CRISPR-Cas systems, and apparatus therefor.

Methods, models, systems, and apparatus for identifying target sequences for Cas enzymes or CRISPR-Cas systems for target sequences and conveying results thereof

The disclosure provides novel large serine recombinases and their respective recognition sites, as well as libraries of orthogonal recombinase recognition sites. Uses of the large serine recombinases, recognition sites, and libraries of orthogonal recombinase recognition sites also are provided.

Recombinases and target sequences

Provided is a modular construction of synthetic gene circuits in mammalian cells using TALE transcriptional repressors. Provided is a method for achieving regulated expression of two proteins: an expression cassette A comprises a feedback element coding sequence, a promoter A, a protein A and TALER protein A encoding gene linked by means of a self-cleaving polypeptide, and a target sequence A (comprising an shRNA1 target sequence); an expression cassette B comprises a feedback element coding sequence, a promoter B, a protein B and TALER protein B encoding gene linked by means of a self-cleaving polypeptide, and a target sequence B (comprising an shRNA2 target sequence); an expression cassette C comprises a constitutive promoter and an activating element-coding sequence; a recombinant vector A having the expression cassette A, a recombinant vector B having the expression cassette B, and a recombinant vector C having the expression cassette C are introduced into host cells to regulate the expression of protein A and protein B by adding shRNA1 or shRNA2.

Modular construction of synthetic gene circuits in mammalian cells using TALE transcriptional repressors

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Cambridge, MA, United States of America

Yinqing Li