@article{a7554b5802254b759f12a43c20cf608b,
title = "Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing",
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",
author = "Nu{\~n}ez, {James K.} and Jin Chen and Pommier, {Greg C.} and Cogan, {J. Zachery} and Replogle, {Joseph M.} and Carmen Adriaens and Ramadoss, {Gokul N.} and Quanming Shi and Hung, {King L.} and Samelson, {Avi J.} and Pogson, {Angela N.} and Kim, {James Y.S.} and Amanda Chung and Leonetti, {Manuel D.} and Chang, {Howard Y.} and Martin Kampmann and Bernstein, {Bradley E.} and Volker Hovestadt and Gilbert, {Luke A.} and Weissman, {Jonathan S.}",
note = "Funding Information: We are grateful to Alex Ge, Matt Laurie, Christina Liem, Chris Hsiung, and Albert Xu for technical assistance and Tessa Bertozzi, Bruce Conklin, Sandy Johnson, Barbara Panning, Fyodor Urnov, and members of the Weissman and Gilbert labs for helpful discussions. This work was funded by DARPA Safe Genes and PREPARE programs (to L.A.G. and J.S.W.), the HHMI Hanna H. Gray Fellows Program (to J.K.N.), the Jane Coffin Childs Memorial Fund (to J.C.), NIH ( K99/R00 GM134154 to J.C., F31 NS115380 to J.M.R., F32 AG063487 to A.J.S., RM1-HG007735 to H.Y.C., DP1CA216873 to B.E.B., 1RM1HG009490 to J.S.W., DP2 CA239597 to L.A.G., and DP2 GM119139 to M.K.), and NSF Graduate Research Fellowship (to G.N.R. and A.C.). L.A.G. is supported by a Goldberg-Benioff Endowed Professorship . J.S.W. and H.Y.C. are HHMI Investigators. B.E.B. is the Bernard and Mildred Kayden Endowed MGH Research Institute Chair, and an American Cancer Society Research Professor. This study was supported in part by UCSF HDFCCC Laboratory for Cell Analysis Shared Resource Facility (NIH P30CA082103 ) and by the Gene Regulation Observatory at the Broad Institute . The IGI supported oligonucleotide pools. Funding Information: We are grateful to Alex Ge, Matt Laurie, Christina Liem, Chris Hsiung, and Albert Xu for technical assistance and Tessa Bertozzi, Bruce Conklin, Sandy Johnson, Barbara Panning, Fyodor Urnov, and members of the Weissman and Gilbert labs for helpful discussions. This work was funded by DARPA Safe Genes and PREPARE programs (to L.A.G. and J.S.W.), the HHMI Hanna H. Gray Fellows Program (to J.K.N.), the Jane Coffin Childs Memorial Fund (to J.C.), NIH (K99/R00 GM134154 to J.C. F31 NS115380 to J.M.R. F32 AG063487 to A.J.S. RM1-HG007735 to H.Y.C. DP1CA216873 to B.E.B. 1RM1HG009490 to J.S.W. DP2 CA239597 to L.A.G. and DP2 GM119139 to M.K.), and NSF Graduate Research Fellowship (to G.N.R. and A.C.). L.A.G. is supported by a Goldberg-Benioff Endowed Professorship. J.S.W. and H.Y.C. are HHMI Investigators. B.E.B. is the Bernard and Mildred Kayden Endowed MGH Research Institute Chair, and an American Cancer Society Research Professor. This study was supported in part by UCSF HDFCCC Laboratory for Cell Analysis Shared Resource Facility (NIH P30CA082103) and by the Gene Regulation Observatory at the Broad Institute. The IGI supported oligonucleotide pools. J.K.N. J.S.W. and L.A.G. led the conception, design, and interpretation of the project as well as writing of the manuscript with the assistance of all of the coauthors. J.K.N. led the design and conducting of the CRISPRoff experiments. G.C.P. and L.A.G. led the design and conducting of the CRISPRon experiments with assistance from A.C. J.C. designed the tiling sgRNA libraries and analyzed screen, RNA-seq, and ChIP-seq datasets. J.Z.C. G.N.R. A.J.S. and M.K. led the iPSC and neuron experiments. J.M.R. designed and cloned the genome-wide sgRNA library with assistance from A.N.P. J.Y.S.K. and M.D.L. constructed the mNG-tagged cell lines. C.A. V.H. and B.E.B. designed, performed, and analyzed the WGBS experiments. Q.S. K.L.H. and H.Y.C. designed, performed, and analyzed the PVT1 enhancer experiments. J.K.N. J.C. G.C.P. L.A.G. and J.S.W. have filed patent applications related to CRISPRoff, CRISPRon, and CRISPRi/a screening. J.M.R. consults for Maze Therapeutics. L.A.G. J.S.W. H.Y.C. and B.E.B. consult for and hold equity in Chroma Medicine. J.S.W. declares outside interest in KSQ Therapeutics, Maze Therapeutics, Amgen, and Tessera Therapeutics. M.K. serves on the Scientific Advisory Boards of Engine Biosciences, Casma Therapeutics, and Cajal Neuroscience. B.E.B. declares outside interests in Fulcrum Therapeutics, Arsenal Biosciences, HiFiBio, and Cell Signaling Technologies. H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, and is an advisor for 10x Genomics, Arsenal Biosciences, and Spring Discovery. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. One or more of the authors of this paper received support from a program designed to increase minority representation in science. Publisher Copyright: {\textcopyright} 2021 Elsevier Inc.",
year = "2021",
month = apr,
day = "29",
doi = "10.1016/j.cell.2021.03.025",
language = "English (US)",
volume = "184",
pages = "2503--2519.e17",
journal = "Cell",
issn = "0092-8674",
publisher = "Cell Press",
number = "9",
}