Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.
Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.|Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.
Pratiksha I
PI
Thakore
Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.|University Program in Genetics and Genomics, Duke University, Durham, North Carolina, USA.
Anthony M
AM
D'Ippolito
Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.|Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA.
Lingyun
L
Song
Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.|Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA.
Alexias
A
Safi
Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
Nishkala K
NK
Shivakumar
Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.|Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.
Ami M
AM
Kabadi
Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.|Department of Biostatistics &Bioinformatics, Duke University Medical Center, Durham, North Carolina, USA.
Timothy E
TE
Reddy
Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.|Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA.
Gregory E
GE
Crawford
Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.|Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.|Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA.
Charles A
CA
Gersbach
D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods*; D015967:Gene Expression Regulation, Viral; D005914:Globins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012097:Repressor Proteins; D014764:Viral Proteins; D005914:Globins