title: Genome-scale CRISPR-Cas9 knockout screening in human cells.
abstract: The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.|McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors: Ophir
authors: O
authors: Shalem
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.|McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors: Neville E
authors: NE
authors: Sanjana
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.
authors: Ella
authors: E
authors: Hartenian
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.|Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA.
authors: Xi
authors: X
authors: Shi
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.|McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors: David A
authors: DA
authors: Scott
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.
authors: Tarjei
authors: T
authors: Mikkelson
authors: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors: Dirk
authors: D
authors: Heckl
authors: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors: Benjamin L
authors: BL
authors: Ebert
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.
authors: David E
authors: DE
authors: Root
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.
authors: John G
authors: JG
authors: Doench
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.|McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors: Feng
authors: F
authors: Zhang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D002470:Cell Survival / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D044842:Cullin Proteins / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D055786:Gene Knockout Techniques; D015723:Gene Library; D016514:Genes, Neurofibromatosis 1; D016515:Genes, Neurofibromatosis 2; D056426:Genetic Loci; D005820:Genetic Testing / Q000379:methods*; D055106:Genome-Wide Association Study; D006801:Humans; D007211:Indoles / Q000627:therapeutic use; D016086:Lentivirus; D056892:Mediator Complex / Q000235:genetics; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics*; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D012641:Selection, Genetic; D013449:Sulfonamides / Q000627:therapeutic use; D014157:Transcription Factors / Q000235:genetics; D000077484:Vemurafenib; D048490:raf Kinases / Q000037:antagonists & inhibitors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C503645:CUL3 protein, human; D044842:Cullin Proteins; D007211:Indoles; C460107:MED12 protein, human; D056892:Mediator Complex; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; C534292:TADA2B protein, human; D014157:Transcription Factors; D000077484:Vemurafenib; D048490:raf Kinases