title: Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.
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.
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Silvana
authors.initials: S
authors.lastname: Konermann
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Brigham
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Alexandro E
authors.initials: AE
authors.lastname: Trevino
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Joung
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Omar O
authors.initials: OO
authors.lastname: Abudayyeh
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Clea
authors.initials: C
authors.lastname: Barcena
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Hsu
authors.affiliation: Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA.
authors.forename: Naomi
authors.initials: N
authors.lastname: Habib
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [5] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Gootenberg
authors.affiliation: 1] Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan [2] JST, PRESTO 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nishimasu
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Nureki
authors.affiliation: 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics; D005820:Genetic Testing; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors; D022661:RNA, Untranslated / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013449:Sulfonamides / Q000494:pharmacology; D015533:Transcriptional Activation / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics
chemical_list: D064130:CRISPR-Associated Proteins; D018076:DNA, Complementary; D007211:Indoles; C528407:PLX 4720; D022661:RNA, Untranslated; D013449:Sulfonamides; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf