CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

Luke A. Gilbert; Matthew H. Larson; Leonardo Morsut; Zairan Liu; Gloria A. Brar; Sandra E. Torres; Noam Stern-Ginossar; Onn Brandman; Evan H. Whitehead; Jennifer A. Doudna; Wendell A. Lim; Jonathan S. Weissman; Lei S. Qi

doi:10.1016/j.cell.2013.06.044

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

Luke A. Gilbert, Matthew H. Larson, Leonardo Morsut, Zairan Liu, Gloria A. Brar, Sandra E. Torres, Noam Stern-Ginossar, Onn Brandman, Evan H. Whitehead, Jennifer A. Doudna, Wendell A. Lim, Jonathan S. Weissman, Lei S. Qi

Department of Molecular Genetics

Weizmann Institute of Science

Research output: Contribution to journal › Article › peer-review

Abstract

The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

Original language	English
Pages (from-to)	442-451
Number of pages	10
Journal	Cell
Volume	154
Issue number	2
DOIs	https://doi.org/10.1016/j.cell.2013.06.044
State	Published - 18 Jul 2013

All Science Journal Classification (ASJC) codes

General Biochemistry,Genetics and Molecular Biology

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@article{61e7b54bd07b4de096aaacc1733228cb,

title = "CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes",

abstract = "The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.",

author = "Gilbert, {Luke A.} and Larson, {Matthew H.} and Leonardo Morsut and Zairan Liu and Brar, {Gloria A.} and Torres, {Sandra E.} and Noam Stern-Ginossar and Onn Brandman and Whitehead, {Evan H.} and Doudna, {Jennifer A.} and Lim, {Wendell A.} and Weissman, {Jonathan S.} and Qi, {Lei S.}",

note = "The authors thank Martin Jinek for discussion and distribution of the dCas9gene. The authors also thank Jason Park, Martin Kampmann, Mike Bassik,and Xin Xiong for lentiviral vectors, technical advice, and helpful discussion.L.S.Q. acknowledges support from the UCSF Center for Systems and Syn-thetic Biology. This work was supported by NIH P50 GM102706 (J.S.W. andJ.A.D.), NIH P50 GM081879 (L.S.Q., W.A.L., Z.L., and E.H.W.), NIH U01CA168370 (J.S.W.), Howard Hughes Medical Institute (L.A.G., G.A.B., O.B.,J.A.D., J.S.W., and W.A.L.), NSF SynBERC EEC-0540879 (W.A.L.), NIGMSIMSD (S.E.T.), the Human Frontiers Scientific Program (N.S. and L.M.), and aRuth L. Kirschstein National Research Service Award (M.H.L.)",

year = "2013",

month = jul,

day = "18",

doi = "10.1016/j.cell.2013.06.044",

language = "الإنجليزيّة",

volume = "154",

pages = "442--451",

journal = "Cell",

issn = "0092-8674",

publisher = "Elsevier B.V.",

number = "2",

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TY - JOUR

T1 - CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

AU - Gilbert, Luke A.

AU - Larson, Matthew H.

AU - Morsut, Leonardo

AU - Liu, Zairan

AU - Brar, Gloria A.

AU - Torres, Sandra E.

AU - Stern-Ginossar, Noam

AU - Brandman, Onn

AU - Whitehead, Evan H.

AU - Doudna, Jennifer A.

AU - Lim, Wendell A.

AU - Weissman, Jonathan S.

AU - Qi, Lei S.

N1 - The authors thank Martin Jinek for discussion and distribution of the dCas9gene. The authors also thank Jason Park, Martin Kampmann, Mike Bassik,and Xin Xiong for lentiviral vectors, technical advice, and helpful discussion.L.S.Q. acknowledges support from the UCSF Center for Systems and Syn-thetic Biology. This work was supported by NIH P50 GM102706 (J.S.W. andJ.A.D.), NIH P50 GM081879 (L.S.Q., W.A.L., Z.L., and E.H.W.), NIH U01CA168370 (J.S.W.), Howard Hughes Medical Institute (L.A.G., G.A.B., O.B.,J.A.D., J.S.W., and W.A.L.), NSF SynBERC EEC-0540879 (W.A.L.), NIGMSIMSD (S.E.T.), the Human Frontiers Scientific Program (N.S. and L.M.), and aRuth L. Kirschstein National Research Service Award (M.H.L.)

PY - 2013/7/18

Y1 - 2013/7/18

N2 - The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

AB - The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

UR - http://www.scopus.com/inward/record.url?scp=84880571335&partnerID=8YFLogxK

U2 - 10.1016/j.cell.2013.06.044

DO - 10.1016/j.cell.2013.06.044

M3 - مقالة

SN - 0092-8674

VL - 154

SP - 442

EP - 451

JO - Cell

JF - Cell

IS - 2

ER -

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

Abstract

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