An enhanced CRISPR repressor for targeted mammalian gene regulation

Nan Cher Yeo; Alejandro Chavez; Alissa Lance-Byrne; Yingleong Chan; David Menn; Denitsa Milanova; Chih-Chung Kuo; Xiaoge Guo; Sumana Sharma; Angela Tung; Ryan J Cecchi; Marcelle Tuttle; Swechchha Pradhan; Elaine T Lim; Noah Davidsohn; Mo R Ebrahimkhani; James J Collins; Nathan E Lewis; Samira Kiani; George M Church

doi:10.1038/s41592-018-0048-5

An enhanced CRISPR repressor for targeted mammalian gene regulation

Nat Methods. 2018 Aug;15(8):611-616. doi: 10.1038/s41592-018-0048-5. Epub 2018 Jul 16.

Authors

Nan Cher Yeo^{1

2}, Alejandro Chavez³, Alissa Lance-Byrne¹, Yingleong Chan^{1

2}, David Menn⁴, Denitsa Milanova^{1

2}, Chih-Chung Kuo^{5

6}, Xiaoge Guo^{1

2}, Sumana Sharma⁷, Angela Tung¹, Ryan J Cecchi¹, Marcelle Tuttle¹, Swechchha Pradhan⁴, Elaine T Lim^{1

2}, Noah Davidsohn^{1

2}, Mo R Ebrahimkhani^{4

8}, James J Collins^{1

9

10

11

12}, Nathan E Lewis^{5

6

13}, Samira Kiani¹⁴, George M Church^{15

16}

Affiliations

¹ Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA.
² Department of Genetics, Harvard Medical School, Boston, MA, USA.
³ Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY, USA. ac4304@cumc.columbia.edu.
⁴ School of Biological and Health Systems Engineering, Ira A. Fulton Schools of Engineering, Arizona State University, Tempe, AZ, USA.
⁵ Department of Bioengineering, University of California, San Diego, San Diego, CA, USA.
⁶ Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, San Diego, CA, USA.
⁷ Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge, UK.
⁸ Division of Gastroenterology and Hematology, Mayo Clinic College of Medicine and Science, Phoenix, AZ, USA.
⁹ Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, MA, USA.
¹⁰ Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA.
¹¹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
¹² Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹³ Department of Pediatrics, University of California, San Diego, San Diego, CA, USA.
¹⁴ School of Biological and Health Systems Engineering, Ira A. Fulton Schools of Engineering, Arizona State University, Tempe, AZ, USA. samira.Kiani@asu.edu.
¹⁵ Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA. gchurch@genetics.med.harvard.edu.
¹⁶ Department of Genetics, Harvard Medical School, Boston, MA, USA. gchurch@genetics.med.harvard.edu.

Abstract

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems*
Gene Expression Regulation*
Gene Silencing*
Genes, Synthetic
HEK293 Cells
Humans
Methyl-CpG-Binding Protein 2 / genetics
Methyl-CpG-Binding Protein 2 / metabolism
RNA, Guide, CRISPR-Cas Systems / genetics
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism
Repressor Proteins / genetics
Repressor Proteins / metabolism

Substances

MECP2 protein, human
Methyl-CpG-Binding Protein 2
RNA, Guide, CRISPR-Cas Systems
Recombinant Fusion Proteins
Repressor Proteins
CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding