ENCODE 3 (2012-2017) expanded production and added new types of assays 8 (Fig. 1, Extended Data Fig. 1), which revealed landscapes of RNA binding and the 3D organization of chromatin via methods such as chromatin interaction analysis by paired-end tagging (ChIA-PET) and Hi-C chromosome conformation capture. Phases 2 and 3 delivered 9,239 experiments (7,495 in human and 1,744 in mouse) in more than 500 cell types and tissues, including mapping of transcribed regions and transcript isoforms, regions of transcripts recognized by RNA-binding proteins, transcription factor binding regions, and regions that harbour specific histone modifications, open chromatin, and 3D chromatin interactions. The results of all of these experiments are available at the ENCODE portal (http://www.encodeproject.org). These efforts, combined with those of related projects and many other laboratories, have produced a greatly enhanced view of the human genome (Fig. 2), identifying 20,225 protein-coding and 37,595 noncoding genes

Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) have an ameliorated clinical phenotype and in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites have been shown to significantly increase expression of endogenous fetal hemoglobin (HbF). To broaden the therapeutic window beyond a single editing approach, we have explored combinations of cis and trans editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of one trans and one cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0 thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe beta globin phenotype.

Highlights d Reactivation of SMARCA4 globally potentiates regulatory DNA accessibility d Promoter chromatin states gate the effects of distal chromatin remodeling d Clustered SMARCA4 occupancy triggers broad remodeling of chromatin domains d Domain remodeling particularly permits high level expression of developmental genes

No abstract

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products.Here we report that the potency and specificity of epigenetic transcriptional repression are linked through promoter keyhole sites, the targeting of which triggers nearcomplete repression with single-gene accuracy. Potent repression, in turn, enables durable programming via reliable induction of mitotically heritable expression programs, offering new potential for engineered cell therapies. 7