Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing

James K Nuñez; Jin Chen; Greg C Pommier; J Zachery Cogan; Joseph M Replogle; Carmen Adriaens; Gokul N Ramadoss; Quanming Shi; King L Hung; Avi J Samelson; Angela N Pogson; James Y S Kim; Amanda Chung; Manuel D Leonetti; Howard Y Chang; Martin Kampmann; Bradley E Bernstein; Volker Hovestadt; Luke A Gilbert; Jonathan S Weissman

doi:10.1016/j.cell.2021.03.025

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing

Cell. 2021 Apr 29;184(9):2503-2519.e17. doi: 10.1016/j.cell.2021.03.025. Epub 2021 Apr 9.

Authors

James K Nuñez¹, Jin Chen¹, Greg C Pommier², J Zachery Cogan³, Joseph M Replogle⁴, Carmen Adriaens⁵, Gokul N Ramadoss⁶, Quanming Shi⁷, King L Hung⁷, Avi J Samelson⁶, Angela N Pogson⁸, James Y S Kim⁹, Amanda Chung¹⁰, Manuel D Leonetti⁹, Howard Y Chang¹¹, Martin Kampmann¹², Bradley E Bernstein⁵, Volker Hovestadt¹³, Luke A Gilbert¹⁴, Jonathan S Weissman¹⁵

Affiliations

¹ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA.
² Department of Urology, University of California, San Francisco, CA 94158, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158, USA.
³ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, CA 94158, USA.
⁴ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, CA 94158, USA; Medical Scientist Training Program, University of California, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA.
⁵ Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA; Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02129, USA.
⁶ Institute for Neurodegenerative Diseases, University of California, San Francisco, CA 94158.
⁷ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA 94305, USA.
⁸ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA.
⁹ Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
¹⁰ Department of Urology, University of California, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, CA 94158, USA.
¹¹ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
¹² Institute for Neurodegenerative Diseases, University of California, San Francisco, CA 94158; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA.
¹³ Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02215, USA.
¹⁴ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Department of Urology, University of California, San Francisco, CA 94158, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158, USA. Electronic address: luke.gilbert@ucsf.edu.
¹⁵ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA. Electronic address: weissman@wi.mit.edu.

Abstract

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

Keywords: CRISPR; DNA methylation; cell therapy; dCas9; epigenetics.

Publication types

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

MeSH terms

CRISPR-Cas Systems*
Cell Differentiation
Cellular Reprogramming*
CpG Islands
DNA Methylation
Epigenesis, Genetic*
Epigenome*
Gene Editing*
Gene Silencing
Histone Code
Humans
Induced Pluripotent Stem Cells / cytology*
Induced Pluripotent Stem Cells / metabolism
Neurons / cytology*
Neurons / metabolism
Protein Processing, Post-Translational

Abstract

Publication types

MeSH terms

Grants and funding