title: Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system.
abstract: Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.
authors: 1] Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA [2] Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors: Albert W
authors: AW
authors: Cheng
authors: Haoyi
authors: H
authors: Wang
authors: Hui
authors: H
authors: Yang
authors: Linyu
authors: L
authors: Shi
authors: Yarden
authors: Y
authors: Katz
authors: Thorold W
authors: TW
authors: Theunissen
authors: Sudharshan
authors: S
authors: Rangarajan
authors: Chikdu S
authors: CS
authors: Shivalila
authors: Daniel B
authors: DB
authors: Dadon
authors: Rudolf
authors: R
authors: Jaenisch
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003850:Deoxyribonuclease I / Q000235:genetics*; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D019076:Transgenes; D058727:RNA, Small Untranslated
chemical_list: D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D003850:Deoxyribonuclease I; D058727:RNA, Small Untranslated
references: Science. 2009 Dec 11;326(5959):1509-12
references: 19933107
references: Nat Biotechnol. 2013 Mar;31(3):227-9
references: 23360964
references: Mol Pharmacol. 2004 Dec;66(6):1361-71
references: 15340042
references: Nat Methods. 2013 Mar;10(3):243-5
references: 23396285
references: Nat Biotechnol. 2013 Sep;31(9):827-32
references: 23873081
references: Nat Methods. 2013 Mar;10(3):239-42
references: 23377379
references: Science. 2009 Dec 11;326(5959):1501
references: 19933106
references: Cell. 2013 Feb 28;152(5):1173-83
references: 23452860
references: Nat Rev Genet. 2012 Sep;13(9):613-26
references: 22868264
references: Science. 2012 Aug 17;337(6096):816-21
references: 22745249
references: Cell. 2009 Aug 21;138(4):722-37
references: 19703398
references: Science. 2013 Feb 15;339(6121):823-6
references: 23287722
references: Science. 2013 Feb 15;339(6121):819-23
references: 23287718
references: Nat Biotechnol. 2013 Mar;31(3):233-9
references: 23360965
references: Cell. 2013 May 9;153(4):910-8
references: 23643243
references: Adv Drug Deliv Rev. 2009 Jul 2;61(7-8):513-26
references: 19394375
references: Nature. 2012 Feb 15;482(7385):331-8
references: 22337052
references: Bioinformatics. 2004 Feb 12;20(3):307-15
references: 14960456
references: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1495-500
references: 10660690
references: Nat Med. 2010 Aug;16(8):903-8
references: 20616797
references: Nat Biotechnol. 2011 Feb;29(2):149-53
references: 21248753
references: Cell. 2013 Jul 18;154(2):442-51
references: 23849981
references: Annu Rev Genet. 2011;45:273-97
references: 22060043
references: Elife. 2013 Jan 29;2:e00471
references: 23386978
references: Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14628-33
references: 9843940