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Results for “Genomics”

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bioRxiv2019

A draft genome sequence of the miniature parasitoid wasp, Megaphragma amalphitanum

Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals to conquer new ecological niches. However, this process is poorly understood at the molecular level. Here, we report genomic and transcriptomic features of arguably the smallest known insect - the parasitoid wasp, Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the genome and transcriptome sizes of this parasitoid wasp are comparable to other members of the Chalcidoidea superfamily. Moreover, the gene content of M. amalphitanum compared to other chalcid wasps is remarkably conserved. Among the very rare cases of apparent gene loss is centrosomin, which encodes an important centrosome component; the absence of this protein might be related to the large number of anucleate neurons in M. amalphitanum. Intriguingly, we also observed significant changes in M. amalphitanum transposable element dynamics over time, whereby an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Thus, while the M. amalphitanum genomic data reveal certain features that may be linked to the unusual biological properties of this organism, miniaturization is not associated with a large decrease in genome complexity.

genomics↗

bioRxiv2019

The genome of the blind soil-dwelling and ancestrally wingless dipluran Campodea augens, a key reference hexapod for studying the emergence of insect innovations

The dipluran two-pronged bristletail Campodea augens is a blind ancestrally wingless hexapod with the remarkable capacity to regenerate lost body appendages such as its long antennae. As sister group to Insecta (sensu stricto), Diplura are key to understanding the early evolution of hexapods and the origin and evolution of insects. Here we report the 1.2-Gbp draft genome of C. augens and results from comparative genomic analyses with other arthropods. In C. augens we uncovered the largest chemosensory gene repertoire of ionotropic receptors in the animal kingdom, a massive expansion which might compensate for the loss of vision. We found a paucity of photoreceptor genes mirroring at the genomic level the secondary loss of an ancestral external photoreceptor organ. Expansions of detoxification and carbohydrate metabolism gene families might reflect adaptations for foraging behaviour, and duplicated apoptotic genes might underlie its high regenerative potential.\n\nThe C. augens genome represents one of the key references for studying the emergence of genomic innovations in insects, the most diverse animal group, and opens up novel opportunities to study the under-explored biology of diplurans.

genomics↗

bioRxiv2019

The genomic diversification of clonally propagated grapevines

Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better understand the consequences of clonal propagation and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03.\n\nThough most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and the increased likelihood of methylated cytosines to spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones.\n\nThese data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clonal propagation is associated with the accumulation of putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions such that mutations are less frequent in coding than noncoding regions of the genome.

genomics↗

bioRxiv2019

Magnus Representation of Genome Sequences

We introduce an alignment-free method, the Magnus Representation, to analyze genome sequences. The Magnus Representation captures higher-order information in genome sequences. We combine our approach with the idea of k-mers to define an effectively computable Mean Magnus Vector. We perform phylogenetic analysis on three datasets: mosquito-borne viruses, filoviruses, and bacterial genomes. Our results on ebolaviruses are consistent with previous phylogenetic analyses, and confirm the modern viewpoint that the 2014 West African Ebola outbreak likely originated from Central Africa. Our analysis also confirms the close relationship between Bundibugyo ebolavirus and Tai Forest ebolavirus. For bacterial genomes, our method is able to classify relatively well at the family and genus level, as well as at higher levels such as phylum level. The bacterial genomes are also separated well into Gram-positive and Gram-negative subgroups.

genomics↗

bioRxiv2019

Genomic and phylogenetic analysis of Salmonella Typhimurium and its monophasic variants responsible for invasive endemic infections in Colombia

Salmonellosis is an endemic human infection, associated with both sporadic cases and outbreaks throughout Colombia. Typhimurium is the most common Colombian serovar of Salmonella enterica, responsible for 32.5% of the Salmonella infections. Whole genome sequencing (WGS) is being used increasingly in Europe and the USA to study the epidemiology of Salmonella, but there has not yet been a WGS-based analysis of Salmonella associated with bloodstream infection in Colombia. Here, we analysed 209 genome sequences of Colombian S. Typhimurium and monophasic S. 4,[5],12:i:-isolates from Colombia from 1999 to 2017. We used a core genome-based maximum likelihood tree to define seven distinct clusters which were predominantly Sequence Type (ST) 19 isolates. We also identified the first ST313 and monophasic ST34 isolates to be reported in Colombia. The history of each cluster was reconstructed with a Bayesian tree to reveal a timeline of evolution. Cluster 7 was closely related to European multidrug-resistant (MDR) DT104. Cluster 4 became the dominant variant of Salmonella in 2016, and resistance to nalidixic acid was associated with a plasmid-encoded qnrB19 gene. Our findings suggest multiple transfers of S. Typhimurium between Europe and Colombia.\n\nAuthor summaryThe large-scale genome sequencing of Salmonella Typhimurium and monophasic Salmonella 4,[5],12:i:-involved bloodstream isolates from Colombia. The two serovars were responsible for about 1/3 of Salmonella infections in Colombia in the past 20 years. To identify the population structure we used Whole Genome Sequencing, performed in silico sequence typing, obtained phylogenetic trees, inferred the evolutionary history, detected the plasmids and prophages, and associated the antibiotic resistance (AMR) genotype with phenotype. Different clusters showed temporal replacement. The Colombian sequence type 313 was distinct from African lineages due to the absence of a key virulence-related gene, bstA. One of the Colombian clusters is likely to belong to the global epidemic of DT104, according to the evolutionary history and the AMR profile. The most common cluster in recent years was resistant to nalidixic acid and carried a plasmid-mediated antibiotic resistant gene qnrB19. Our findings will inform the ongoing efforts to combat Salmonellosis by Colombian public health departments.

genomics↗

bioRxiv2019

The Genomics of Selfing in Maize (Zea mays ssp. mays): Catching Purging in the Act

In plants, self-fertilization is both an important reproductive strategy and a valuable genetic tool. In theory, selfing increases homozygosity at a rate of 0.50 per generation. Increased homozygosity can uncover recessive deleterious variants and lead to inbreeding depression, unless it is countered by the loss of these variants by genetic purging. Here we investigated the dynamics of purging on genomic scale by testing three predictions. The first was that heterozygous, putatively deleterious SNPs were preferentially lost from the genome during continued selfing. The second was that the loss of deleterious SNPs varied as a function of recombination rate, because recombination increases the efficacy of selection by uncoupling linked variants. Finally, we predicted that genome size (GS) decreases during selfing, due to the purging of deleterious transposable element (TE) insertions. We tested these three predictions by following GS and SNP variants in a series of selfed maize (Zea mays ssp. mays) lines over six generations. In these lines, putatively deleterious alleles were purged, and purging was more pronounced in highly recombining regions. Homozygosity increased more slowly than expected; instead of increasing by 50% each generation, it increased by 35% to 40%. Finally, three lines showed dramatic decreases in GS, losing an average of 398 Mb from their genomes over the short timeframe of our experiment. TEs were the principal component of loss, and GS loss was more likely for lineages that began with more TE and more chromosomal knob repeats. Overall, this study documented remarkable GS loss - as much DNA as three Arabidopsis thaliana genomes, on average - in only a few generations of selfing.

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bioRxiv2019

Identifying TCDD-resistance genes via murine and rat comparative genomics and transcriptomics

The aryl hydrocarbon receptor (AHR) mediates many of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone is insufficient to explain the widely different outcomes among organisms. Attempts to identify unknown factor(s) have been confounded by genetic variability of model organisms. Here, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS), as well as C57BL/6 and DBA/2 mice. We supplement these with whole-genome sequencing and transcriptomic analyses of the corresponding rat models: Long-Evans (L-E) and Han/Wistar (H/W) rats. These integrated multi-species genomic and transcriptomic data were used to identify genes associated with TCDD-response phenotypes.\n\nWe identified several genes that show consistent transcriptional changes in both transgenic mice and rats. Hepatic Pxdc1 was significantly repressed by TCDD in C57BL/6, rWT mice, and in L-E rat. Three genes demonstrated different AHRE-1 (full) motif occurrences within their promoter regions: Cxxc5 had fewer occurrences in H/W, as compared with L-E; Sugp1 and Hgfac (in either L-E or H/W respectively). These genes also showed different patterns of mRNA abundance across strains.\n\nThe AHR isoform explains much of the transcriptional variability: up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (30% and 10% unique to DEL and rWT respectively following 500 g/kg TCDD). Genomic and transcriptomic evidence allowed identification of genes potentially involved in phenotypic outcomes: Pxdc1 had differential mRNA abundance by phenotype; Cxxc5 had altered AHR binding sites and differential mRNA abundance.\n\nAuthor SummaryEnvironmental contaminants such as dioxins cause many toxic responses, anything from chloracne (common in humans) to death. These toxic responses are mostly regulated by the Ahr, a ligand-activated transcription factor with roles in drug metabolism and immune responses, however other contributing factors remain unclear. Studies are complicated by the underlying genetic heterogeneity of model organisms. Our team evaluated a number of mouse and rat models, including two strains of mouse, two strains of rat and three transgenic mouse lines which differ only at the Ahr locus, that present widely different sensitivities to the most potent dioxin: 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). We identified a number of changes to gene expression that were associated with different toxic responses. We then contrasted these findings with results from whole-genome sequencing of the H/W and L-E rats and found some key genes, such as Cxxc5 and Mafb, which might contribute to TCDD toxicity. These transcriptomic and genomic datasets will provide a valuable resource for future studies into the mechanisms of dioxin toxicities.

genomics↗

bioRxiv2019

Genome-wide Associations of Flavivirus Capsid Proteins

Dengue virus (DENV) and Zika virus (ZIKV) are both positive sense single-stranded RNA viruses. They are packaged within the virion with a capsid (C) protein to form the nucleocapsid. Based on cryo-electron microscopy imaging, the nucleocapsid has been described as lacking symmetry, whilst there is distinguishable separation of the C proteins from the viral RNA (vRNA) genome. Here, to elucidate the architecture of the nucleocapsid of DENV serotype 2 and ZIKV, we used a nuclease digestion assay and next-generation sequencing to map the respective vRNA genome wide association with the C protein in vitro. The C protein exhibited non-uniform binding along the vRNA, and as C protein concentration increased, the normalized read counts also increased. A saturation point of 1:100 (vRNA:C protein monomers) was found, and binding regions showed variable saturation patterns. We also observed that C protein had a preference for G-rich sequences for both viruses. Taken together, we demonstrate that the DENV 2 and ZIKV C proteins bind vRNA in a non-uniform manner with distinct patterns of association.\n\nSingificance StatementOur study demonstrates that flavivirus capsid proteins associate with the viral genome at specific sites rather than in a uniform manner as commonly expected. We estimate the number of capsid proteins binding to a single genomic RNA. We proceed to locate the capsid binding sites along the viral genomes of Dengue and Zika viruses. We characterize the binding sites in terms of affinity and analyze the nucleotide composition and sequence motifs at binding sites. We cross-reference binding sites against SHAPE reactivity data corresponding to local RNA secondary structure, which allows us to identify structural motifs of capsid binding sites. As capsid proteins are essential for viral packaging, these interactions may form attractive targets for therapeutic intervention.

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bioRxiv2019

CNCC: An analysis tool to determine genome-wide DNA break end structure at single-nucleotide resolution

DNA double-stranded breaks (DSBs) are potentially deleterious events in a cell. The end structures (blunt, 3- and 5-overhangs) at sites of double-stranded breaks contribute to the fate of their repair and provide critical information for consequences of the damage. Here, we describe the use of a coverage-normalized cross correlation analysis (CNCC) to process high-precision genome-wide break mapping data, and determine genome-wide break end structure distributions at single-nucleotide resolution. For the first time, on a genome-wide scale, our analysis revealed the increase in the 5 to 3 end resection following etoposide treatment, and the global progression of the resection due to the removal of DNA topoisomerase II cleavage complexes. Further, our method distinguished the change in the pattern of DSB end structure with increasing doses of the drug. The ability of this method to determine DNA break end structures without a priori knowledge of break sequences or genomic position should have broad applications in understanding genome instability.

genomics↗

bioRxiv2019

The Chinese chestnut genome: a reference for species restoration

Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.

genomics↗

bioRxiv2019

Identification of Pathogenic Structural Variants in Rare Disease Patients through Genome Sequencing

PurposeClinical whole genome sequencing is becoming more common for determining the molecular diagnosis of rare disease. However, standard clinical practice often focuses on small variants such as single nucleotide variants and small insertions/deletions. This leaves a wide range of larger \"structural variants\" that are not commonly analyzed in patients.\n\nMethodsWe developed a pipeline for processing structural variants for patients who received whole genome sequencing through the Undiagnosed Diseases Network (UDN). This pipeline called structural variants, stored them in an internal database, and filtered the variants based on internal frequencies and external annotations. The remaining variants were manually inspected and then interesting findings were reported as research variants to clinical sites in the UDN.\n\nResultsOf 477 analyzed UDN cases, 286 cases ({approx} 60%) received at least one structural variant as a research finding. The variants in 16 cases ({approx} 4%) are considered \"Certain\" or \"Highly likely\" molecularly diagnosed and another 4 cases are currently in review. Of those 20 cases, at least 13 were identified originally through our pipeline with one finding leading to identification of a new disease. As part of this paper, we have also released the collection of variant calls identified in our cohort along with heterozygous and homozygous call counts. This data is available at https://github.com/HudsonAlpha/UDN_SV_export.\n\nConclusionStructural variants are key genetic features that should be analyzed during routine clinical genomic analysis. For our UDN patients, structural variants helped solve {approx} 4% of the total number of cases ({approx} 13% of all genome sequencing solves), a success rate we expect to improve with better tools and greater understanding of the human genome.

genomics↗

bioRxiv2019

Genomic sequencing of the aquatic Fusarium spp. QHM and BWC1 and their potential application in environmental protection

Fusarium species are distributed widely in ecosystems of a wide pH range and play a pivotal role in the aquatic community through the degradation of xenobiotic compounds and secretion of secondary metabolites. The elucidation of their genome would therefore be highly impactful with regard to the control of environmental pollution. Therefore, in this study, two indigenous strains of aquatic Fusarium, QHM and BWC1, were isolated from a coal mine pit and a subterranean river respectively, cultured under acidic conditions, and sequenced. Phylogenetic analysis of these two isolates was conducted based on the sequences of internal transcript (ITS1 and ITS4) and encoding {beta}-microtubulin (TUB2), translation elongation factors (TEFs) and the second large sub-unit of RNA polymerase (RPB2). Fusarium, QHM could potentially represent a new species within the Fusarium fujikuroi species complex. Fusarium BWC1 were found to form a clade with Fusarium subglutinans NRRL 22016, and predicted to be Fusarium subglutinans. Shot-gun sequencing on the Illumina Hiseqx10 Platform was used to elucidate the draft genomes of the two species. Gene annotation and functional analyses revealed that they had bio-degradation pathways for aromatic compounds; further, their main pathogenic mechanism was found to be the efflux pump. To date, the genomes of only a limited number of acidic species from the Fusarium fujikuroi species complex, especially from the aquatic species, have been sequenced. Therefore, the present findings are novel and have important potential for the future in terms of environmental control.\n\nIMPORTANCEFusarium genus has over 300 species and were distributed in a variety of ecosystem. Increasing attention has been drawn to Fusarium due to the importance in aquatic community, pathogenicity and environmental protection. The genomes of the strains in this work isolated in acidic condition, were sequenced. The analysis has indicated that the isolates were able to biodegrade xenobiotics, which makes it potentially function as environmental bio-agent for aromatic pollution control and remediation. Meanwhile, the virulence and pathogenicity were also predicted for reference of infection control. The genome information may lay foundation for the fungal identification, disease prevention resulting from these isolates and other \"-omics\" research. The isolates were phylogenetically classified into Fusarium fujikuroi species complex by means of concatenated gene analysis, serving as new addition to the big complex.

genomics↗

bioRxiv2019

Aquila: diploid personal genome assembly and comprehensive variant detection based on linked reads

Variant discovery in personal, whole genome sequence data is critical for uncovering the genetic contributions to health and disease. We introduce a new approach, Aquila, that uses linked-read data for generating a high quality diploid genome assembly, from which it then comprehensively detects and phases personal genetic variation. Assemblies cover >95% of the human reference genome, with over 98% in a diploid state. Thus, the assemblies support detection and accurate genotyping of the most prevalent types of human genetic variation, including single nucleotide polymorphisms (SNPs), small insertions and deletions (small indels), and structural variants (SVs), in all but the most difficult regions. All heterozygous variants are phased in blocks that can approach arm-level length. The final output of Aquila is a diploid and phased personal genome sequence, and a phased VCF file that also contains homozygous and a few unphased heterozygous variants. Aquila represents a cost-effective evolution of whole-genome reconstruction that can be applied to cohorts for variation discovery or association studies, or to single individuals with rare phenotypes that could be caused by SVs or compound heterozygosity.

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bioRxiv2019

Chromosome level draft genomes of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), an alien invasive pest in China

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) is a severely destructive pest native to the Americas, but has now become an alien invasive pest in China, and causes significant economic loss. Therefore, in order to make effective management strategies, it is highly essential to understand genomic architecture and its genetic background. In this study, we assembled two chromosome scale genomes of the fall armyworm, representing one male and one female individual procured from Yunnan province of China. The genome sizes were identified as 542.42 Mb with N50 of 14.16 Mb, and 530.77 Mb with N50 of 14.89 Mb for the male and female FAW, respectively. We predicted about 22,201 genes in the male genome. We found the expansion of cytochrome P450 and glutathione s-transferase gene families, which are functionally related to the intensified detoxification and pesticides tolerance. Further population analyses of corn strain (C strain) and rice strain (R strain) revealed that the Chinese fall armyworm was most likely invaded from Africa. These strain information, genome features and possible invasion source described in this study will be extremely important for making effective strategies to manage the fall armyworms.

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bioRxiv2019

Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information

BackgroundLong DNA reads produced by single molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short read DNA fragments. For de novo assembly, PacBio and Oxford Nanopore Technologies (ONT) are favorite options. However, PacBios SMRT sequencing is expensive for a full human genome assembly and costs over 40,000 USD for 30x coverage as of 2019. ONT PromethION sequencing, on the other hand, is one-twelfth the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBios SMRT sequencing in relation to the quality.\n\nFindingsWe performed whole genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64x coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mbp and a total genome length of 2.8 Gbp. It was comparable to a KOREF assembly constructed using PacBio at 62x coverage (188 Gbp, 2,695 contigs and N50s of 17.9 Mbp). When we applied Hi-C-derived long-range mapping data, an even higher quality assembly for the 64x coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mbp.\n\nConclusionThe pore-based PromethION approach provides a good quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths and is more cost-effective than PacBio at comparable quality measurements.

genomics↗

bioRxiv2019

Insights into human genetic variation and population history from 929 diverse genomes

Genome sequences from diverse human groups are needed to understand the structure of genetic variation in our species and the history of, and relationships between, different populations. We present 929 high-coverage genome sequences from 54 diverse human populations, 26 of which are physically phased using linked-read sequencing. Analyses of these genomes reveal an excess of previously undocumented private genetic variation in southern and central Africa and in Oceania and the Americas, but an absence of fixed, private variants between major geographical regions. We also find deep and gradual population separations within Africa, contrasting population size histories between hunter-gatherer and agriculturalist groups in the last 10,000 years, a potentially major population growth episode after the peopling of the Americas, and a contrast between single Neanderthal but multiple Denisovan source populations contributing to present-day human populations. We also demonstrate benefits to the study of population relationships of genome sequences over ascertained array genotypes. These genome sequences are freely available as a resource with no access or analysis restrictions.

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bioRxiv2019

A classification framework for Bacillus anthracis defined by global genomic structure

Bacillus anthracis, the causative agent of anthrax, is a considerable global health threat affecting wildlife, livestock, and the general public. In this study whole-genome sequence analysis of over 350 B. anthracis isolates was used to establish a new high-resolution global genotyping framework that is both biogeographically informative, and compatible with multiple genomic assays. The data presented in this study shed new light on the diverse global dissemination of this species and indicate that many lineages may be uniquely suited to the geographic regions in which they are found. In addition, we demonstrate that plasmid genomic structure for this species is largely consistent with chromosomal population structure, suggesting vertical inheritance in this bacterium has contributed to its evolutionary persistence. This classification methodology is the first based on population genomic structure for this species and has potential use for local and broader institutions seeking to understand both disease outbreak origins and recent introductions. In addition, we provide access to a newly developed genotyping script as well as the full whole genome sequence analyses output for this study, allowing future studies to rapidly employ and append their data in the context of this global collection. This framework may act as a powerful tool for public health agencies, wildlife disease laboratories, and researchers seeking to utilize and expand this classification scheme for further investigations into B. anthracis evolution.

genomics↗

bioRxiv2019

Concurrent Genome and Epigenome Editing by CRISPR-Mediated Sequence Replacement

Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double stranded break (DSB), followed by NHEJ or HDR mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, i.e. to drive replacement of the DNA sequence intervening the dual cuts via NHEJ. In a proof-of-concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event, and opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

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