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Article ; Online: Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

Thakore, Pratiksha I / Gersbach, Charles A

Methods in molecular biology (Clifton, N.J.)

2016 Volume 1338, Page(s) 71–88

Abstract: Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and ... ...

Abstract	Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.
MeSH term(s)	DNA-Binding Proteins/chemistry ; DNA-Binding Proteins/genetics ; Gene Targeting/methods ; Genetic Engineering/methods ; Genetic Vectors ; Genomics/methods ; HEK293 Cells ; Humans ; Promoter Regions, Genetic ; Synthetic Biology ; Trans-Activators/chemistry ; Trans-Activators/genetics
Chemical Substances	DNA-Binding Proteins ; Trans-Activators
Language	English
Publishing date	2016
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-2932-0_7
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Editing the epigenome: technologies for programmable transcription and epigenetic modulation.

Thakore, Pratiksha I / Black, Joshua B / Hilton, Isaac B / Gersbach, Charles A

Nature methods

2016 Volume 13, Issue 2, Page(s) 127–137

Abstract: Gene regulation is a complex and tightly controlled process that defines cell identity, health and disease, and response to pharmacologic and environmental signals. Recently developed DNA-targeting platforms, including zinc finger proteins, transcription ...

Abstract	Gene regulation is a complex and tightly controlled process that defines cell identity, health and disease, and response to pharmacologic and environmental signals. Recently developed DNA-targeting platforms, including zinc finger proteins, transcription activator-like effectors (TALEs) and the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, have enabled the recruitment of transcriptional modulators and epigenome-modifying factors to any genomic site, leading to new insights into the function of epigenetic marks in gene expression. Additionally, custom transcriptional and epigenetic regulation is facilitating refined control over cell function and decision making. The unique properties of the CRISPR-Cas9 system have created new opportunities for high-throughput genetic screens and multiplexing targets to manipulate complex gene expression patterns. This Review summarizes recent technological developments in this area and their application to biomedical challenges. We also discuss remaining limitations and necessary future directions for this field.
MeSH term(s)	Animals ; CRISPR-Cas Systems ; Epigenesis, Genetic/physiology ; Epigenomics ; Gene Expression Regulation ; Genetic Engineering/methods ; Transcription, Genetic/physiology
Language	English
Publishing date	2016-02
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	2169522-2
ISSN	1548-7105 ; 1548-7091
ISSN (online)	1548-7105
ISSN	1548-7091
DOI	10.1038/nmeth.3733
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Article: Systematically characterizing the roles of E3-ligase family members in inflammatory responses with massively parallel Perturb-seq.

bioRxiv : the preprint server for biology

2023

Abstract: E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the ... ...

Abstract	E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the balance of DC1, DC2, migratory DC and macrophage states and a gradient of DC maturation. Family members grouped into co-functional modules that were enriched for physical interactions and impacted specific programs through substrate transcription factors. E3s and their adaptors co-regulated the same processes, but partnered with different substrate recognition adaptors to impact distinct aspects of the DC life cycle. Genetic interactions were more prevalent within than between modules, and a deep learning model, comβVAE, predicts the outcome of new combinations by leveraging modularity. The E3 regulatory network was associated with heritable variation and aberrant gene expression in immune cells in human inflammatory diseases. Our study provides a general approach to dissect gene function.
Language	English
Publishing date	2023-01-24
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.01.23.525198
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Article ; Online: Mostly natural sequencing-by-synthesis for scRNA-seq using Ultima sequencing.

Simmons, Sean K / Lithwick-Yanai, Gila / Adiconis, Xian / Oberstrass, Florian / Iremadze, Nika / Geiger-Schuller, Kathryn / Thakore, Pratiksha I / Frangieh, Chris J / Barad, Omer / Almogy, Gilad / Rozenblatt-Rosen, Orit / Regev, Aviv / Lipson, Doron / Levin, Joshua Z

Nature biotechnology

2022 Volume 41, Issue 2, Page(s) 204–211

Abstract: Here we introduce a mostly natural sequencing-by-synthesis (mnSBS) method for single-cell RNA sequencing (scRNA-seq), adapted to the Ultima genomics platform, and systematically benchmark it against current scRNA-seq technology. mnSBS uses mostly natural, ...

Abstract	Here we introduce a mostly natural sequencing-by-synthesis (mnSBS) method for single-cell RNA sequencing (scRNA-seq), adapted to the Ultima genomics platform, and systematically benchmark it against current scRNA-seq technology. mnSBS uses mostly natural, unmodified nucleotides and only a low fraction of fluorescently labeled nucleotides, which allows for high polymerase processivity and lower costs. We demonstrate successful application in four scRNA-seq case studies of different technical and biological types, including 5' and 3' scRNA-seq, human peripheral blood mononuclear cells from a single individual and in multiplex, as well as Perturb-Seq. Benchmarking shows that results from mnSBS-based scRNA-seq are very similar to those using Illumina sequencing, with minor differences in results related to the position of reads relative to annotated gene boundaries, owing to single-end reads of Ultima being closer to gene ends than reads from Illumina. The method is thus compatible with state-of-the-art scRNA-seq libraries independent of the sequencing technology. We expect mnSBS to be of particular utility for cost-effective large-scale scRNA-seq projects.
MeSH term(s)	Humans ; Gene Expression Profiling/methods ; Sequence Analysis, RNA/methods ; Leukocytes, Mononuclear ; Single-Cell Gene Expression Analysis ; Single-Cell Analysis/methods ; Nucleotides
Chemical Substances	Nucleotides
Language	English
Publishing date	2022-09-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/s41587-022-01452-6
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Zs.A 2167: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Publisher Correction: Gut CD4

Kiner, Evgeny / Willie, Elijah / Vijaykumar, Brinda / Chowdhary, Kaitavjeet / Schmutz, Hugo / Chandler, Jodie / Schnell, Alexandra / Thakore, Pratiksha I / LeGros, Graham / Mostafavi, Sara / Mathis, Diane / Benoist, Christophe

Nature immunology

2021 Volume 22, Issue 5, Page(s) 666–668

Language	English
Publishing date	2021-02-25
Publishing country	United States
Document type	Published Erratum
ZDB-ID	2016987-5
ISSN	1529-2916 ; 1529-2908
ISSN (online)	1529-2916
ISSN	1529-2908
DOI	10.1038/s41590-021-00916-2
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Zs.A 5294: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MG 42: Show issues

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Article ; Online: Gut CD4

Nature immunology

2021 Volume 22, Issue 2, Page(s) 216–228

Abstract: ... ...

Abstract	CD4
MeSH term(s)	Animals ; Bacteria/immunology ; Bacteria/pathogenicity ; Bacterial Infections/genetics ; Bacterial Infections/immunology ; Bacterial Infections/metabolism ; Bacterial Infections/microbiology ; CD4-Positive T-Lymphocytes/immunology ; CD4-Positive T-Lymphocytes/metabolism ; CD4-Positive T-Lymphocytes/microbiology ; CD4-Positive T-Lymphocytes/parasitology ; Chromatin/genetics ; Chromatin/metabolism ; Citrobacter rodentium/immunology ; Citrobacter rodentium/pathogenicity ; Colon/immunology ; Colon/metabolism ; Colon/microbiology ; Colon/parasitology ; Cytokines/genetics ; Cytokines/metabolism ; Disease Models, Animal ; Gastrointestinal Microbiome ; Gene Expression Profiling ; Heligmosomatoidea/immunology ; Heligmosomatoidea/pathogenicity ; Host-Pathogen Interactions ; Interferon Regulatory Factors/genetics ; Interferon Regulatory Factors/metabolism ; Intestinal Diseases, Parasitic/genetics ; Intestinal Diseases, Parasitic/immunology ; Intestinal Diseases, Parasitic/metabolism ; Intestinal Diseases, Parasitic/parasitology ; Male ; Mice, Inbred C57BL ; Mice, Transgenic ; Nematospiroides dubius/immunology ; Nematospiroides dubius/pathogenicity ; Nippostrongylus/immunology ; Nippostrongylus/pathogenicity ; Phenotype ; Salmonella enterica/immunology ; Salmonella enterica/pathogenicity ; Single-Cell Analysis ; Transcription Factor AP-1/genetics ; Transcription Factor AP-1/metabolism ; Transcriptome ; Mice
Chemical Substances	Chromatin ; Cytokines ; Interferon Regulatory Factors ; Transcription Factor AP-1
Language	English
Publishing date	2021-01-18
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2016987-5
ISSN	1529-2916 ; 1529-2908
ISSN (online)	1529-2916
ISSN	1529-2908
DOI	10.1038/s41590-020-00836-7
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Zs.A 5294: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MG 42: Show issues

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Article ; Online: RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.

Thakore, Pratiksha I / Kwon, Jennifer B / Nelson, Christopher E / Rouse, Douglas C / Gemberling, Matthew P / Oliver, Matthew L / Gersbach, Charles A

Nature communications

2018 Volume 9, Issue 1, Page(s) 1674

Abstract: CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor ( ... ...

Abstract	CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9
MeSH term(s)	Animals ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; CRISPR-Cas Systems ; Cholesterol/blood ; Endonucleases/genetics ; Endonucleases/metabolism ; Gene Silencing ; Genetic Therapy ; Liver/enzymology ; Male ; Mice ; Mice, Inbred C57BL ; Proprotein Convertase 9/blood ; Proprotein Convertase 9/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Staphylococcus aureus/enzymology ; Staphylococcus aureus/genetics ; Transcription, Genetic
Chemical Substances	Bacterial Proteins ; RNA, Guide, CRISPR-Cas Systems ; Cholesterol (97C5T2UQ7J) ; Endonucleases (EC 3.1.-) ; Pcsk9 protein, mouse (EC 3.4.21.-) ; Proprotein Convertase 9 (EC 3.4.21.-)
Language	English
Publishing date	2018-04-26
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-018-04048-4
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Article ; Online: Stem-like intestinal Th17 cells give rise to pathogenic effector T cells during autoimmunity.

Schnell, Alexandra / Huang, Linglin / Singer, Meromit / Singaraju, Anvita / Barilla, Rocky M / Regan, Brianna M L / Bollhagen, Alina / Thakore, Pratiksha I / Dionne, Danielle / Delorey, Toni M / Pawlak, Mathias / Meyer Zu Horste, Gerd / Rozenblatt-Rosen, Orit / Irizarry, Rafael A / Regev, Aviv / Kuchroo, Vijay K

Cell

2021 Volume 184, Issue 26, Page(s) 6281–6298.e23

Abstract: While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 ... ...

Abstract	While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1
MeSH term(s)	Animals ; Autoimmunity ; Cell Movement ; Clone Cells ; Encephalomyelitis, Autoimmune, Experimental/immunology ; Granulocyte-Macrophage Colony-Stimulating Factor/metabolism ; Homeostasis ; Humans ; Interferon-gamma/metabolism ; Interleukin-17/metabolism ; Intestines/immunology ; Mice, Inbred C57BL ; Organ Specificity ; RNA/metabolism ; RNA-Seq ; Receptors, Antigen, T-Cell/metabolism ; Receptors, CXCR6/metabolism ; Receptors, Interleukin/metabolism ; Reproducibility of Results ; Signaling Lymphocytic Activation Molecule Family/metabolism ; Single-Cell Analysis ; Spleen/metabolism ; Stem Cells/metabolism ; Th17 Cells/immunology ; Mice
Chemical Substances	CXCR6 protein, human ; IL23R protein, human ; Interleukin-17 ; Receptors, Antigen, T-Cell ; Receptors, CXCR6 ; Receptors, Interleukin ; SLAMF6 protein, human ; Signaling Lymphocytic Activation Molecule Family ; RNA (63231-63-0) ; Interferon-gamma (82115-62-6) ; Granulocyte-Macrophage Colony-Stimulating Factor (83869-56-1)
Language	English
Publishing date	2021-12-06
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	187009-9
ISSN	1097-4172 ; 0092-8674
ISSN (online)	1097-4172
ISSN	0092-8674
DOI	10.1016/j.cell.2021.11.018
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Zs.A 1167: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Massively parallel phenotyping of coding variants in cancer with Perturb-seq.

Nature biotechnology

2022 Volume 40, Issue 6, Page(s) 896–905

Abstract: Genome sequencing studies have identified millions of somatic variants in cancer, but it remains challenging to predict the phenotypic impact of most. Experimental approaches to distinguish impactful variants often use phenotypic assays that report on ... ...

Abstract	Genome sequencing studies have identified millions of somatic variants in cancer, but it remains challenging to predict the phenotypic impact of most. Experimental approaches to distinguish impactful variants often use phenotypic assays that report on predefined gene-specific functional effects in bulk cell populations. Here, we develop an approach to functionally assess variant impact in single cells by pooled Perturb-seq. We measured the impact of 200 TP53 and KRAS variants on RNA profiles in over 300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison with orthogonal assays. We discovered that KRAS variants did not merely fit into discrete functional categories, but spanned a continuum of gain-of-function phenotypes, and that their functional impact could not have been predicted solely by their frequency in patient cohorts. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, with potential applications in multiple disease settings.
MeSH term(s)	Chromosome Mapping ; Humans ; Lung Neoplasms/genetics ; Phenotype ; Proto-Oncogene Proteins p21(ras)/genetics
Chemical Substances	Proto-Oncogene Proteins p21(ras) (EC 3.6.5.2)
Language	English
Publishing date	2022-01-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/s41587-021-01160-7
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Zs.A 2167: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Author Correction: Massively parallel phenotyping of coding variants in cancer with Perturb-seq.

Nature biotechnology

2022 Volume 40, Issue 11, Page(s) 1691

Language	English
Publishing date	2022-10-18
Publishing country	United States
Document type	Published Erratum
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/s41587-022-01495-9
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Zs.A 2167: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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