Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered...

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Published in:Nature (London) Vol. 517; no. 7536; pp. 583 - 588
Main Authors: Konermann, Silvana, Brigham, Mark D., Trevino, Alexandro E., Joung, Julia, Abudayyeh, Omar O., Barcena, Clea, Hsu, Patrick D., Habib, Naomi, Gootenberg, Jonathan S., Nishimasu, Hiroshi, Nureki, Osamu, Zhang, Feng
Format: Journal Article
Language:English
Published: London Nature Publishing Group UK 29.01.2015
Nature Publishing Group
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ISSN:0028-0836, 1476-4687, 1476-4687
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Abstract Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology. The CRISPR-Cas9 system, a powerful tool for genome editing, has been engineered to activate endogenous gene transcription specifically and potently on a genome-wide scale and applied to a large-scale gain-of-function screen for studying melanoma drug resistance. CRISPR-Cas9 used for gene-expression regulation The CRISPR-Cas9 system has emerged as a powerful tool for genome editing and transcriptional regulation of specific genes. Feng Zhang and colleagues have successfully modified the system to specifically and potently activate endogenous gene transcription on a genome-wide scale, such that it can be used for large-scale functional genomics screens. Application to a genome-wide screen of melanoma cells for genes which when overexpressed can confer resistance to a BRAF inhibitor demonstrates the feasibility of such screens, and also led to the discovery of potential new resistance mechanisms.
AbstractList Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology. The CRISPR-Cas9 system, a powerful tool for genome editing, has been engineered to activate endogenous gene transcription specifically and potently on a genome-wide scale and applied to a large-scale gain-of-function screen for studying melanoma drug resistance. CRISPR-Cas9 used for gene-expression regulation The CRISPR-Cas9 system has emerged as a powerful tool for genome editing and transcriptional regulation of specific genes. Feng Zhang and colleagues have successfully modified the system to specifically and potently activate endogenous gene transcription on a genome-wide scale, such that it can be used for large-scale functional genomics screens. Application to a genome-wide screen of melanoma cells for genes which when overexpressed can confer resistance to a BRAF inhibitor demonstrates the feasibility of such screens, and also led to the discovery of potential new resistance mechanisms.
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology. The CRISPR-Cas9 system, a powerful tool for genome editing, has been engineered to activate endogenous gene transcription specifically and potently on a genome-wide scale and applied to a large-scale gain-of-function screen for studying melanoma drug resistance. CRISPR-Cas9 used for gene-expression regulation The CRISPR-Cas9 system has emerged as a powerful tool for genome editing and transcriptional regulation of specific genes. Feng Zhang and colleagues have successfully modified the system to specifically and potently activate endogenous gene transcription on a genome-wide scale, such that it can be used for large-scale functional genomics screens. Application to a genome-wide screen of melanoma cells for genes which when overexpressed can confer resistance to a BRAF inhibitor demonstrates the feasibility of such screens, and also led to the discovery of potential new resistance mechanisms.
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. We describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We use these engineered Cas9 activation complexes to investigate sgRNA targeting rules for effective transcriptional activation, demonstrate multiplexed activation of 10 genes simultaneously, and upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesize a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes which, upon activation, confer resistance to a BRAF inhibitor. Expected and potentially novel resistance genes are enriched in the top hits and are validated using individual sgRNA as well as cDNA overexpression. The signature of our top screening hits is significantly correlated with gene expression data from clinical melanoma samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.
Audience Academic
Author Konermann, Silvana
Trevino, Alexandro E.
Habib, Naomi
Nishimasu, Hiroshi
Nureki, Osamu
Joung, Julia
Abudayyeh, Omar O.
Barcena, Clea
Brigham, Mark D.
Gootenberg, Jonathan S.
Hsu, Patrick D.
Zhang, Feng
AuthorAffiliation 1 Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA
4 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
2 McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
5 Department of Systems Biology, Harvard Medical School, Boston, MA 02115
7 JST, PRESTO, 2-11-16 Yayoi, Bunkyo, Tokyo, 113-0032, Japan
6 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo, 113-0032, Japan
AuthorAffiliation_xml – name: 2 McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
– name: 6 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo, 113-0032, Japan
– name: 7 JST, PRESTO, 2-11-16 Yayoi, Bunkyo, Tokyo, 113-0032, Japan
– name: 5 Department of Systems Biology, Harvard Medical School, Boston, MA 02115
– name: 4 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
– name: 1 Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA
– name: 3 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Author_xml – sequence: 1
  givenname: Silvana
  surname: Konermann
  fullname: Konermann, Silvana
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Department of Biological Engineering, Massachusetts Institute of Technology
– sequence: 2
  givenname: Mark D.
  surname: Brigham
  fullname: Brigham, Mark D.
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Department of Biological Engineering, Massachusetts Institute of Technology
– sequence: 3
  givenname: Alexandro E.
  surname: Trevino
  fullname: Trevino, Alexandro E.
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Department of Biological Engineering, Massachusetts Institute of Technology
– sequence: 4
  givenname: Julia
  surname: Joung
  fullname: Joung, Julia
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, Department of Biological Engineering, Massachusetts Institute of Technology
– sequence: 5
  givenname: Omar O.
  surname: Abudayyeh
  fullname: Abudayyeh, Omar O.
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Department of Biological Engineering, Massachusetts Institute of Technology
– sequence: 6
  givenname: Clea
  surname: Barcena
  fullname: Barcena, Clea
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Department of Biological Engineering, Massachusetts Institute of Technology
– sequence: 7
  givenname: Patrick D.
  surname: Hsu
  fullname: Hsu, Patrick D.
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Department of Biological Engineering, Massachusetts Institute of Technology
– sequence: 8
  givenname: Naomi
  surname: Habib
  fullname: Habib, Naomi
  organization: Broad Institute of MIT and Harvard, 75 Ames Street
– sequence: 9
  givenname: Jonathan S.
  surname: Gootenberg
  fullname: Gootenberg, Jonathan S.
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Department of Biological Engineering, Massachusetts Institute of Technology, Department of Systems Biology, Harvard Medical School
– sequence: 10
  givenname: Hiroshi
  surname: Nishimasu
  fullname: Nishimasu, Hiroshi
  organization: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan, JST, PRESTO 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan
– sequence: 11
  givenname: Osamu
  surname: Nureki
  fullname: Nureki, Osamu
  organization: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan
– sequence: 12
  givenname: Feng
  surname: Zhang
  fullname: Zhang, Feng
  email: zhang@broadinstitute.org
  organization: Broad Institute of MIT and Harvard, 75 Ames Street, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Department of Biological Engineering, Massachusetts Institute of Technology
BackLink https://www.ncbi.nlm.nih.gov/pubmed/25494202$$D View this record in MEDLINE/PubMed
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Snippet Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe...
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. We describe structure-guided...
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proquest
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Index Database
Enrichment Source
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StartPage 583
SubjectTerms 45/23
631/337/572/2102
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats - genetics
CRISPR-Associated Proteins - genetics
CRISPR-Associated Proteins - metabolism
CRISPR-Cas Systems - genetics
Crystal structure
DNA, Complementary - biosynthesis
DNA, Complementary - genetics
Drug resistance
Drug Resistance, Neoplasm - drug effects
Drug Resistance, Neoplasm - genetics
Enzymes
Epigenetics
Gene expression
Gene Expression Regulation, Neoplastic - genetics
Gene Library
Genetic aspects
Genetic engineering
Genetic Engineering - methods
Genetic Loci - genetics
Genetic regulation
Genetic research
Genetic Testing
Genetic transcription
Genome, Human - genetics
Genomes
Genomics
Health aspects
Humanities and Social Sciences
Humans
Indoles - pharmacology
Melanoma
Melanoma - drug therapy
Melanoma - genetics
multidisciplinary
Nucleotide sequence
Proteins
Proto-Oncogene Proteins B-raf - antagonists & inhibitors
Reproducibility of Results
RNA, Untranslated - biosynthesis
RNA, Untranslated - genetics
RNA, Untranslated - metabolism
Science
Sulfonamides - pharmacology
Transcription factors
Transcriptional Activation - genetics
Up-Regulation - genetics
Title Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
URI https://link.springer.com/article/10.1038/nature14136
https://www.ncbi.nlm.nih.gov/pubmed/25494202
https://www.proquest.com/docview/1651539733
https://www.proquest.com/docview/1652425801
https://pubmed.ncbi.nlm.nih.gov/PMC4420636
Volume 517
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