Endogenous Elements Research Articles

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is widely used for biomolecular mapping but has not been extensively applied for direct element imaging. Established techniques such as LA-ICP-MS and SIMS provide high sensitivity for elemental analysis but lack the ability to simultaneously map elements and biomolecules. Here, we demonstrate the feasibility of MALDI MSI for direct spatial element profiling across multiple biological contexts. We optimized MALDI MSI parameters─including laser power, ionization conditions, and mass resolving power─to enable the detection of endogenous and exogenous elements such as Fe, Ca, Gd, Pt, and Cl. Using a combination of high-resolution FTICR-MS and timsTOF MSI, we assessed ionization efficiency, spectral fidelity, and isotopic accuracy. The method was applied to murine developmental models, genetic metal accumulation disorders, and platinum-based chemotherapy distributions in tumors. Our results demonstrate that MALDI MSI enables element detection while preserving spatial integrity. Computational modeling and spectral similarity analysis confirmed the reliability of isotopologue distributions. These findings establish MALDI MSI as a viable alternative for high-resolution element imaging, offering a complementary approach to LA-ICP-MS and SIMS by integrating atomic and biomolecular spatial distributions. This method expands the analytical capabilities of MALDI MSI and opens new avenues for metallomics, pharmacokinetics, and disease biomarker research.

The ability to precisely control gene expression using small-molecule drugs is a valuable tool in research and has important therapeutic potential. However, existing systems are often limited by the toxicity of the drugs and the need to alter gene sequences or endogenous regulatory elements. Here, we introduce Cyclone (acyclovir-controlled poison exon), an acyclovir-controlled poison exon cassette that can be used for small-molecule control of both transgene and endogenous gene expression. Cyclone is a portable 'intron-poison exon-intron' element that can be inserted into nearly any gene and is completely removed upon acyclovir treatment, leaving the native transcript intact. Cyclone offers tunable, reversible gene expression with nearly undetectable background and a ~295-fold activation. We also present Pac-Cyclone, a cassette that simplifies the generation of cell lines with acyclovir-controlled endogenous gene expression. Finally, we demonstrate the programmability of Cyclone, underscoring its potential for developing diverse genetic circuits controlled by various ligands.

It is unclear how CRISPR editing outcomes vary across the genome and whether undesirable events such as structural variants (SVs) are predictable or preventable. To define a genome-wide map of editability, we performed whole-genome sequencing on 1875 budding yeast clones edited across 16 chromosomes by CRISPR-Cas9 and donor-templated repair. We found that unintended edits, including short indels and SVs, were enriched in specific genomic and sequence contexts. We developed a predictive model, SCORE (System for CRISPR Outcome and Risk Evaluation), which revealed 4.8% of the genome as SV prone, consisting of 562 SV hotspots. Donor repair-enhancing strategies suppressed SV formation in regions with moderate, but not high, predicted risk. Applying SCORE to the Sc2.0 synthetic yeast genome revealed a markedly altered SV landscape due to the removal of endogenous repetitive elements and the insertion of loxP sites. Our study provides the genome-scale map of SV hotspots after CRISPR editing and predictive and experimental tools to mitigate their formation.

Evolution of a melanoma that escapes allogeneic rejection.

Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms. However, these approaches lack direct, scalable readouts of chromatin accessibility across long single-molecule chromatin fibers. Here we leverage double-stranded DNA cytidine deaminases to profile chromatin accessibility at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). With high sequence coverage at targeted loci, TDAC-seq can be integrated with CRISPR perturbations to link genetic edits and their effects on chromatin accessibility on the same single chromatin fiber at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34+ hematopoietic stem and progenitor cells (HSPCs) during erythroid differentiation as well as in pooled CRISPR and base-editing screens tiling an enhancer controlling the globin locus. We further scaled the method to interrogate 947 variants in a GFI1B-linked enhancer associated with myeloproliferative neoplasm risk in a single pooled CRISPR experiment in CD34+ HSPCs. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.

Understanding human embryogenesis by building programmable stem cell-based models.

The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. The main advantage of this system compared to existing techniques is maintenance of endogenous regulatory elements. We used this system to identify both cell-autonomous and non-cell-autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.

Insertion sequence (IS) elements are key drivers of bacterial genome plasticity, yet the overall regulation of their transposition remains poorly understood. This is especially true for the multiple-layer regulation at the donor site, which has been largely overlooked. Using multiple mutation assays, genetic manipulations and reporter genes, this study focuses on characterizing how endogenous DNA sequences, transcriptional and translational factors, and genomic context regulate IS1 transposition from its donor site. Out of six elements within the chromosome of E. coli strain BW25113, IS1A and IS1E (both with the consensus sequence) contribute to over 99.9% of the overall IS1 transposition within the genome while the other four elements without the non-consensus sequence are essentially incapable of transposing. Inducing a ribosomal -1 frameshift at the A6C motif increases transposition over 1000-fold, but this enhancement is largely reversed by restoring InsA-mediated transcriptional regulation. Strikingly, genomic sequences flanking IS1 elements appreciably modulate transposition by promoting transcription or facilitating formation of transpososomes, a phenomenon that remains under-studied. Finally, IS1 was confirmed to undergo replicative transposition intramolecularly, a mechanism shown here to be independent of transposase levels in the cell. These findings contribute to our understanding of mobile genetic element regulation and potentially offer strategies for mitigating their potentially harmful effects.

SummaryMammalian hosts deploy a multitude of germline-encoded mechanisms to detect and restrict virus infection. These must avoid pathological responses to endogenous retroviruses (ERVs) and other endogenous retrotransposable elements (RTEs) – viruses and virus-like genomic parasites that have invaded the host germline and are passed down the generations as host genes. Although the location, specificity and sensitivity of innate pattern-recognition receptors (PRRs) and restriction factors are tuned to facilitate discrimination of infecting viruses from those that are part of self, immune cross-reactions do occur. The RTE viral heritage may therefore compromise the ability of the host to respond to virus infection without risking pathology. Nevertheless, it also provides opportunities for RTEs to be co-opted as an alarm amplification system, repurposed as antiviral factors and contribute to the evolution of antiviral genes.

Researchers in various fields continue to discover improved ways of local delivery of drugs to specific locations and try to increase the efficiency of these methods. Extensive research has been done on smart nano-biomaterials for drug delivery systems (DDS) in different dimensions. With the advancement of biomedical nanotechnology, conventional smart DDS with stimuli- responsive capability has been developed. Smart nano-biomaterials can respond to environmental changes caused by endogenous or exogenous elements: endogenous factors such as environmental pH, temperature gradient, enzymes, oxidation, and reduction potential. As well as exogenous factors, including light radiation, ultrasound, electric and magnetic fields. Currently, smart DDSs count as a major category in DDS and disease treatment. Currently, smart DDS are of great interest in drug delivery and treatment of diseases. With the improvements in gene and protein therapy, new methods have been presented to treat diseases without effective conventional treatment, especially cancer. Finally, the use of nanoparticles expanded due to the need for appropriate gene and protein delivery systems. This review discusses the advantages of protein and gene therapy, their challenges, and gene and protein delivery systems with nanoparticle-based delivery.

Abstract Background: The tremendous success of chimeric antigen receptor-T (CAR-T) cell therapy in haematological malignancies has not been recapitulated in solid tumours, owing to tumour-induced immunosuppression, tumour heterogeneity and inefficient tumour trafficking. One promising solution includes “armouring” CAR-T cells with therapeutic transgenes. Indeed, we demonstrated that CAR-T cells engineered to express dendritic cell growth factor Flt3L could effectively engage host anti-tumour immunity crucial for overcoming antigen-negative relapse1. However, synthetic promoters have demonstrated insufficiencies in driving tumour-restricted cytokine expression, which had caused systemic toxicities and trial termination2. The advent of CRISPR/Cas9 gene-editing tool has enabled the precise engineering of CAR-T cells for safety and efficacy enhancements. We previously showed that CRISPR/Cas9-mediated knock-out (KO) of immunosuppressive gene A2AR enhanced CAR-T cell function3. Now, we aim to exploit a CRISPR/Cas9-mediated knock-in (KI) strategy to leverage endogenous gene regulatory elements to restrict transgene expression to tumour for enhanced safety and efficacy. Methods: Genome-wide RNA sequencing was performed on CAR-T cells isolated from tumours and spleens of mice. 27 genes upregulated in intratumoural relative to splenic CAR-T cells were identified as potential KI sites. As KI disrupts target gene expression, the impact of each gene KO on CAR-T cell function/phenotype was first assessed. 7 genes without adverse impact following KO had GFP knocked in. Results: NR4A2 and RGS16 emerged as tumour-specific promoters upon KI. While NR4A2 was highly tumour-restricted and could deliver highly toxic cytokines (e.g., IL-12) without inducing toxicities in mice, RGS16 had high intratumoural expression and could mediate the efficacy of less potent cytokines (e.g., IL-2). Conclusions: Endogenous tumour-specific promoters enabled the generation of IL-12- and IL-2-expressing CAR-T cells with enhanced safety and efficacy in syngeneic and xenogeneic mouse models that was concomitant with improved CAR-T cell polyfunctionality and activation of host anti-tumour immunity. Notably, this CRISPR-KI strategy was applicable using patient-derived CAR-T cells, demonstrating its clinical translatability. Citation Format: Kah Min Yap, Amanda X. Y. Chen, Imran G. House, Phillip K. Darcy and Paul A. Beavis. Identifying Optimal Tumour-specific Promoters for CRISPR/Cas9 Engineering of Armoured CAR T Cells with Enhanced Safety and Efficacy [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr LT01.

EFSA was requested by the European Commission to provide a scientific opinion on new developments in biotechnology, including new genomic techniques, as applied to animals for food, feed and other agricultural uses. A horizon‐scanning exercise identified a variety of animals obtained with new genomic techniques, with the potential to reach the EU market in the short, medium and long term. No novel hazards have been identified that are linked to either the modification process or the newly introduced trait, when SDN‐1, SDN‐2 and comparable techniques (e.g. base editing or prime editing) were compared to established genomic techniques (EGTs) or conventional breeding. Hazards posed by SDN‐3 are of the same nature as those posed by EGTs and the targeted insertion may reduce the potential hazards associated with the disruption of endogenous genes and/or regulatory elements in the recipient genome. Hazards posed by the new trait resulting from the introduced transgenic or intragenic DNA sequence are of the same nature as those posed by EGTs. Hazards posed by the new trait resulting from the introduced cisgenic DNA sequence are of the same nature as those posed by conventional breeding. Off‐target mutations from genome editing are similar in nature to those from conventional breeding and do not pose novel hazards. Consequently, based on the currently available data, no new potential hazards, and thus, no new risks to humans, animals or the environment have been identified. A thorough evaluation of existing EFSA guidance documents for the risk assessment of GM animals revealed that their principles and recommendations provide the basis for assessing the risks of new genomic technique (NGT) animals for food, feed and other agricultural uses; however, the current texts cover only partially some areas (e.g. animal health and welfare) and may need updates, adaptations or enhancements on a case‐by‐case basis to fully address NGT‐related risks.

Leaf senescence is an essential physiological process which is accompanied by the transfer and recycling of nutrients from aging organs to growing tissues. This intricate process is finely orchestrated by multiple endogenous and exogenous elements, including leaf age, environmental signals, and developmental cues. Plant small peptides, serve as intercellular signaling molecules, which regulate processes like stem cell homeostasis, stress responses, immune responses, pollen-stigma recognition, and organ abscission. Recent studies revealed that small peptides are important regulators during leaf senescence progression. For example, CLE14 and CLE42 peptides function in the suppression of leaf senescence though modulating ROS and ethylene signals, respectively. SCOOP10 and SCOOP12 peptides, however, modulate leaf senescence via MIK2 receptor-like kinases in an antagonistic manner. In this review, we conclude recent research in modulating leaf senescence by small peptides, encompassing both positive and negative regulators. Our aim is to provide a deeper comprehension of the diverse signaling pathways engaging in leaf senescence.

Endogenous viral elements (EVEs) serve as molecular fossils that record the ancient co-evolutionary arms race between viruses and their hosts. In this study, by analyzing 105 host crustacean genomes, we identified 252 infectious hypodermal and haematopoietic necrosis virus-derived EVEs (IHHNV-EVEs), which include 183 ancient and 6 recently inserted EVEs. These IHHNV-EVEs are widely distributed among Decapoda, Thoracica, and Isopoda, with some of them exhibiting a syntenic distribution relative toneighboringhost sequences, suggesting that the IHHNV or its ancestor are potential pathogens of these species with a long-time dynamic interaction during the evolutionary history. An expansion of IHHNV-EVEs was observed indecapodagenomes, reflecting a reinforced arm race betweendecapodaand IHHNV. Notably, we found that nearly all recent IHHNV-EVEs were laboratory contaminants, except for a single authentic integration in Penaeus monodon that persists intact across 16 samples from the 2 populations. These temporal dynamics-ancient genomic stabilization versus modern colonization activity-highlight that EVEs serve as dual archives: historical records of past conflicts and active participants in current evolutionary battles. Our findings redefine viral genomic colonization as a continuum, where ancient EVE fixation coexists with persistent integration processes, providing new insights into host-virus co-evolutionary trajectories.

Geminiviruses constitute a diverse group of plant viruses with small, circular single-stranded DNA genomes. While most geminiviruses possess monopartite genomes, the genus Begomovirus uniquely includes both monopartite and bipartite members. The evolutionary origin of the second component of begomovirus (DNA-B) has been a subject of considerable debate. Two primary hypotheses propose that DNA-B originated from a modified monopartite genome or through the capture of a satellite DNA. Recent discoveries of unclassified bipartite geminiviruses call for a reevaluation of these hypotheses. To address this, we investigated the evolutionary history of the begomovirus nuclear shuttle protein (NSP) through homolog searches, comparative genomics, and structural protein analyses. Our findings unambiguously demonstrated that NSP is homologous to the coat protein (CP) but originated from a CP encoded by an ancient geminivirus lineage, distinct from begomoviruses. This ancient lineage is represented by bipartite viruses integrated into plant genomes of the genus Rhododendron. These results challenge the prevailing paradigm regarding the evolutionary origin of NSP and offer new insights into the evolution of begomovirus genome architecture.

Endogenous viral elements (EVEs) are genomic sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express co-opted proteins in their host. Furthermore, some EVEs that are expressed as proteins have become part of cellular genes that are fusions of hosts and EVE sequences. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and some of them contain ORFs and can be expressed as proteins. We have shown that an EPV containing an ORF is part of the guinea pig gene enRep-M9l. This gene is broadly transcribed in vivo, indicating that it can be translated into a protein. By generating antibodies against the enRep coding sequence of the enRep-M9l ORF, we showed that the protein enRep-M9l is expressed in vivo and in the guinea pig-derived cell line JH4. By immunofluorescence and in situ proximity ligation assays, we observed that enRep-M9l protein has a cytoplasmic localization near microtubules. The results of this study suggest that the guinea pig EPV-derived protein enRep-M9l is a microtubule-associated protein. To our knowledge, this is the second demonstration that an EPV-derived protein is expressed in vivo.

Characterization of Aspergillus oryzae mutant and its application in heterologous lipase expression.

Transcriptomics of HERVs reveals clinico-biological characterization of LTR5_Hs and HERVS71 loci in gastric cancer.

Self-organization of mouse embryonic stem cells into reproducible pre-gastrulation embryo models via CRISPRa programming.

Tailoring capsid-directed evolution technology for improved AAV-mediated CAR-T generation.