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Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications., Competing Interests: DECLARATION OF INTERESTS J.K.J. has, or had during the course of this research, financial interests in several companies developing gene-editing or epigenetic-editing technology: Beam Therapeutics, Blink Therapeutics, Chroma Medicine, Editas Medicine, EpiLogic Therapeutics, ETx Inc., Excelsior Genomics, Hera Biolabs, Monitor Biotechnologies, Pairwise Plants, Poseida Therapeutics, SeQure Dx Inc., and Verve Therapeutics. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. J.K.J. is a consultant for Beam Therapeutics, Chroma Medicine, ETx Inc., Pairwise Plants, SeQure Dx Inc., and Verve Therapeutics and is a scientific co-founder of these companies and of Blink Therapeutics, Editas Medicine, EpiLogic Therapeutics, Excelsior Genomics, and Monitor Biotechnologies. G.B., J.K.J., M.N.R., and Y.E.T. are inventors on a patent application that encompasses work described in this paper. J.K.J. and Y.E.T. are inventors on patents or patent applications describing various gene- and epigenetic-editing technologies. M.N.R. receives research support from ACD and Merck Serono for work unrelated to this study. S.I. and S.P.G. are employees of Verve Therapeutics. J.K.J. is a member of the Advisory Board of Cell Genomics.

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The 32-item Drinking Behavior Interview (DBI) was administered to 103 patients newly admitted to a private, inpatient, alcohol problems treatment unit. When intercorrelations among the 32 variables were subjected to factor analysis, DBI profiles of alcoholics in this population differed primarily along four major dimensions: close interpersonal relationships, physical symptoms, societal functioning and alcohol consumption. Whereas the DBI has previously been used to define a single composite alcoholism index, these results suggest the importance of considering differences in components of the DBI profile pattern.