BackgroundGenetically modified organisms (GMOs) have numerous biomedical, agricultural and environmental applications. Development of accurate methods for the detection of GMOs is a prerequisite for the identification and control of authorized and unauthorized release of these engineered organisms into the environment and into the food chain. Current detection methods are unable to detect uncharacterized GMOs, since either the DNA sequence of the transgene or the amino acid sequence of the protein must be known for DNA-based or immunological-based detection, respectively.MethodsHere we describe the application of an epigenetics-based approach for the detection of mammalian GMOs via analysis of chromatin structural changes occurring in the host nucleus upon the insertion of foreign or endogenous DNA.ResultsImmunological methods combined with DNA next generation sequencing enabled direct interrogation of chromatin structure and identification of insertions of various size foreign (human or viral) DNA sequences, DNA sequences often used as genome modification tools (e.g. viral sequences, transposon elements), or endogenous DNA sequences into the nuclear genome of a model animal organism.ConclusionsThe results provide a proof-of-concept that epigenetic approaches can be used to detect the insertion of endogenous and exogenous sequences into the genome of higher organisms where the method of genetic modification, the sequence of inserted DNA, and the exact genomic insertion site(s) are unknown.General significanceMeasurement of chromatin dynamics as a sensor for detection of genomic manipulation and, more broadly, organism exposure to environmental or other factors affecting the epigenomic landscape are discussed.

BackgroundIdentification of wound-specific markers would represent an important step toward damaged tissue detection and targeted delivery of biologically important materials to injured sites. Such delivery could minimize the amount of therapeutic materials that must be administered and limit potential collateral damage on nearby normal tissues. Yet, biological markers that are specific for injured tissue sites remain elusive.MethodsIn this study, we have developed an immunohistological approach for identification of protein epitopes specifically exposed in wounded tissue sites.ResultsUsing ex-vivo tissue samples in combination with fluorescently-labeled antibodies we show that actin, an intracellular cytoskeletal protein, is specifically exposed upon injury. The targetability of actin in injured sites has been demonstrated in vivo through the specific delivery of anti-actin conjugated particles to the wounded tissue in a lethal rat model of grade IV liver injury.ConclusionsThese results illustrate that identification of injury-specific protein markers and their targetability for specific delivery is feasible.General significanceIdentification of wound-specific targets has important medical applications as it could enable specific delivery of various products, such as expression vectors, therapeutic drugs, hemostatic materials, tissue healing, or scar prevention agents, to internal sites of penetrating or surgical wounds regardless of origin, geometry or location.

There is an established relationship between primary DNA sequence, secondary and tertiary chromatin structure, and transcriptional activity, suggesting that observed differences in one of these properties may reflect changes in the others. Here, we exploit these relationships to show that variations in DNA structure can be used to identify a wide range of genomic alterations in mammalian samples. In this proof-of-concept study we characterized and compared genome-wide histone occupancy by ChIP-Seq, DNA accessibility by ATAC-Seq, and chromosomal conformation by Hi-C for five CRISPR/Cas9-modified mammalian cell lines and their unmodified parent strains, as well as in one modified tissue sample and its parent strain. The results showed that the impact of genomic alterations on each of the levels of DNA organization varied depending on mutation type (insertion or deletion), size, and genomic location. The largest genomic alterations we identified included chromosomal rearrangements and deletions (greater than 200 Kb) in four of the modified cell lines, which can be difficult to identify by standard whole genome sequencing analysis. This multi-level DNA organizational analysis provides a sensitive approach for identifying a wide range of genomic and epigenomic perturbations that can be utilized for biomedical and biosecurity applications.