Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.
Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy.
Angelo
A
Amabile
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy.
Alessandro
A
Migliara
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
Paola
P
Capasso
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
Mauro
M
Biffi
Center for Translational Genomics and Bioinformatics, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
Davide
D
Cittaro
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. Electronic address: naldini.luigi@hsr.it.
Luigi
L
Naldini
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. Electronic address: lombardo.angelo@hsr.it.
Angelo
A
Lombardo
D020134:Catalytic Domain; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D053595:Embryonic Stem Cells / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism
C000717967:DNMT3A protein, human; D051741:Kruppel-Like Transcription Factors; D012097:Repressor Proteins; C109484:ZNF10 protein, human; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A
B2M-null cells; CRISPR/Cas9; DNA methylation; DNMT3L; KRAB-ZFP/KAP1; TALE; TET1; epigenetic editing; gene therapy; permanent gene silencing
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