Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming.

Summary Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

SUMMARY The type II bacterial CRISPR/Cas system is a novel genome engineering technology with the ease of multiplexed gene targeting. Here we created reporter and conditional mutant mice by co-injection of zygotes with Cas9 mRNA, different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2 and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.