Many human diseases have an underlying genetic component. The development and application of methods to prevent the inheritance of damaging mutations through the human germline could have significant health benefits, and currently include preimplantation genetic diagnosis and carrier screening. Ma et al. take this a step further by attempting to remove a disease mutation from the human germline through gene editing 1 . They assert the following advances: (i) the correction of a pathogenic gene mutation responsible for hypertrophic cardiomyopathy in human embryos using CRISPR-Cas9 and (ii) the avoidance of mosaicism in edited embryos. In the case of correction, the authors conclude that repair using the homologous chromosome was as or more frequent than mutagenic nonhomologous end-joining (NHEJ). Their conclusion is significant, if validated, because such a "self-repair" mechanism would allow gene correction without the introduction of a repair template. While the authors' analyses relied on the failure to detect mutant alleles, here we suggest approaches to provide direct evidence for interhomologue recombination and discuss other events consistent with the data. We also review the biological constraints on inter-homologue recombination in the early embryo.In their first approach, Ma et al. used donor sperm from a patient heterozygous for the MYBPC3 ΔGAGT mutation to fertilize wild-type oocytes, such that half of the embryos started out as wild type at the MYBPC3 locus and half heterozygous. Fertilized zygotes were injected with Cas9 and an sgRNA directed to create a double-strand break (DSB) in the mutant paternal allele. The authors report that 24% of the embryos at day 3 of development were mosaic, with some cells of the embryo containing the mutant paternal locus, either intact or modified by NHEJ, together with a wild-type locus. Remaining cells of the embryo contained only a detectable wild-type allele. While some zygotes were also co-injected with a wild-type, exogenous, single-stranded oligodeoxynucleotide template (ssODN) with two synonymous mutations, no mutations consistent with ssODN-templated repair were detected. Furthermore, 'wild-type only' cells were present at a similar frequency both in the presence and absence of the ssODN. The authors infer that these cells arose by homology-directed repair (HDR) of the mutant paternal allele using the wild-type maternal allele as a template, i.e., inter-homologue recombination, leading to gene correction.In a second approach, earlier, MII-phase oocytes were coinjected with Cas9 complexes and donor sperm. In this case, mosaicism was not detected, except in a single embryo, which contained both 'wildtype only' cells and ones heterozygous for wild-type and ssODN-templated alleles. Although wild-type embryos were expected at 50% frequency, they appeared to comprise 72% of embryos.
Transcriptional enhancers are a predominant class of noncoding regulatory elements that activate cell type-specific gene expression. Tissue-specific enhancer-associated chromatin signatures have proven useful to identify candidate enhancer elements at a genome-wide scale, but their sensitivity for the comprehensive detection of all enhancers active in a given tissue in vivo remains unclear. Here we show that a substantial proportion of in vivo enhancers are hidden from discovery by conventional chromatin profiling methods. In an initial comparison of over 1,200 in vivo validated tissue-specific enhancers with tissue-matched mouse developmental epigenome data, 14% (n=286) of active enhancers did not show canonical enhancer-associated chromatin signatures in the tissue in which they are active. To assess the prevalence of enhancers not detectable by conventional chromatin profiling approaches in more detail, we used a high throughput transgenic enhancer reporter assay to systematically screen over 1.3 Mb of mouse genomic sequence at two critical developmental loci, assessing a total of 281 consecutive 5kb regions for in vivo enhancer activity in mouse embryos. We observed reproducible enhancer-reporter activity in 88 tissue-specific elements, 26% of which did not show canonical enhancer-associated chromatin signatures in the corresponding tissues. Overall, we find these hidden enhancers are indistinguishable from marked enhancers based on levels of evolutionary conservation, enrichment of transcription factor families, and genomic positioning relative to putative target genes. In combination, our retrospective and prospective studies assessed only 0.1% of the mouse genome and identified 309 tissue-specific enhancers that are hidden from current chromatin-based enhancer identification approaches. Our findings suggest the existence of tens of thousands of active enhancers throughout the genome that remain undetected by current chromatin profiling approaches and are an unappreciated source of additional genome function of import in interpreting growing whole human genome sequencing data.
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