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.
James K Nuñez  1 ,  Jin Chen  1 ,  Greg C Pommier  2 ,  J Zachery Cogan  3 ,  Joseph M Replogle  4 ,  Carmen Adriaens  5 ,  Gokul N Ramadoss  6 ,  Quanming Shi  7 ,  King L Hung  7 ,  Avi J Samelson  6 ,  Angela N Pogson  8 ,  James Y S Kim  9 ,  Amanda Chung  10 ,  Manuel D Leonetti  9 ,  Howard Y Chang  11 ,  Martin Kampmann  12 ,  Bradley E Bernstein  5 ,  Volker Hovestadt  13 ,  Luke A Gilbert  14 ,  Jonathan S Weissman  15
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: [email protected].
  • 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: [email protected].
PMID: 33838111 DOI: 10.1016/j.cell.2021.03.025
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.