Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery
Authors: Berkley E Gryder
Jian Jin
Tiffany A Wang
Dongbo Lu
Javed Khan
Silvia Pomella
Nathaniel A Hathaway
Xufen Yu
Anna M Chiarella
Kyle V Butler
Journal: Nature biotechnology (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Gene expression; CRISPR-Cas9 systems; chemical epigenetic modifiers; dose-dependent activation; target genes
Affiliations: Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, Chapel Hill, NC, USA., Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA., Oncogenomics Section, Genetics Branch, National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD, USA., Department of Pediatric Hematology and Oncology, Bambino Gesu' Children's Hospital, Rome, Italy., Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. ., Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, Chapel Hill, NC, USA. .

Abstract:  Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome. 

Retracted: No

PubPeer comments:
 dCas9 still has [residual endonuclease] , and one also has to consider that u201c[mismatches at dCas9 binding sites can be as high as ten] u201d This study is no different - here are some sequences (there are many others) which integrate the Cas9 to the human genome, and that too in different locations (maybe off-targets, maybe it was different gRNAs used) That can only happen if there is a DSB. SRR10154186.17775972.1 17775972 TGAGCACCTCAGAGGCAGGCAGTCCCAAGGCTTCAGCACAGAGTCTATCCTGCCCAAGAGGAACAGCGACAAGCTGATCG SRR8860489.11030553.1 11030553 TTTCTAGAGAATAAAGTTTGCTAGAAAGAGAAGAAAAGATCTTATCTCAGGAAGCTGCTGCCGCACGAGAGCTTTCAGCA SRR8860489.25574080.1 25574080 TTTCTAGAGAATAAAGTTTGCTAGAAAGAGAAGAAAAGATCTTATCTCAGGCAGCTGCTGCCGCACGAGAGCTTTCAGCA ,