Details:

Alternative Title:
Nature
Personal Author:
Konermann, Silvana ; Brigham, Mark D. ; Trevino, Alexandro E. ; Joung, Julia ; Abudayyeh, Omar O. ; ... More +
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 Less -
Description:
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.
Subject:
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Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Proteins
CRISPR-Cas Systems
DNA, Complementary
Drug Resistance, Neoplasm
Gene Expression Regulation, Neoplastic
Gene Library
Genetic Engineering
Genetic Loci
Genetic Testing
Genome, Human
Humans
Indoles
Melanoma
Proto-Oncogene Proteins B-raf
Reproducibility Of Results
RNA, Untranslated
Sulfonamides
Transcriptional Activation
Up-Regulation

Source:
Nature. 517(7536):583-588.
Document Type:
Journal Article
Funding:
DP1 MH100706/MH/NIMH NIH HHS/United States ; DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; R01 NS062849/NS/NINDS NIH HHS/United States ; R01 NS073124/NS/NINDS NIH HHS/United States ; R01-NS07312401/NS/NINDS NIH HHS/United States
DP1 MH100706/MH/NIMH NIH HHS/United States ; DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; R01 NS062849/NS/NINDS NIH HHS/United States ; R01 NS073124/NS/NINDS NIH HHS/United States ; R01-NS07312401/NS/NINDS NIH HHS/United States Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:00240015f8e83fdc18d2356f7aaa2060e230d14be0f83ff74a6b8bda785ee102
File Type:

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