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PubMed2026

Interfering with DNA repair pathways to enhance CRISPR-Cas9-mediated homology-directed repair in a chelicerate genetic model.

The two-spotted spider mite, Tetranychus urticae, is a major pest and an emerging genetic model. Recent CRISPR-Cas9 advances, especially the SYNCAS method for maternal delivery of Cas9 ribonucleoproteins, have enabled precise genome editing in this and other difficult-to-transform arthropods. Yet SYNCAS-mediated knockins vary in efficiency, possibly due to competition between DNA repair pathways, whose mechanisms in T. urticae and other chelicerates remain unknown. Here, we provide the first functional analysis of double-strand break repair in a chelicerate. Loss of DNA polymerase theta (Polθ) redirects repair almost entirely toward homology-directed repair, whereas absence of Ligase IV has no detectable impact. Using a reporter assay targeting phytoene desaturase, we demonstrate that Polθ-deficient strains enhance incorporation of repair templates, even when mutations are distant from the cut site. Also, insertion of larger fragments is improved. Finally, disrupting Polθ imposes only a modest fitness cost, highlighting its value for future genome engineering in this species.

Acari↗

PubMed2026

Artificial intelligence-driven advancements in agricultural biotechnology.

The need for faster and more informative data processing for better decision-making is driving the adoption of artificial intelligence (AI) in the agricultural sector. Thanks to recent advancements in computer science and the increase in computational powers of modern computers, AI is not only augmenting traditional solutions, but also helping in developing novel solutions to existing challenging matters. AI-driven models have an exceptional ability to identify patterns and combine a diverse collection of data together and make inference. The increasing pressure on farmlands posed by the growing global population and climate change is lessening growth, yield, and productivity ultimately posing risk to food security worldwide. Incorporation of AI in agriculture has the potential to drive farming efficiency to new heights. This comprehensive review critically evaluates the evolution of AI in agricultural biotechnology from a theoretical concept to a global phenomenon. A comprehensive literature search was performed using major scientific databases, including PubMed, Web of Science, Embase, Scopus, Lens and the Cochrane Library. In this review, we empirically demonstrate the fields advancement toward more capable AI systems and discuss the current applications of AI across crop improvement and precision agriculture such as crop improvement and genetic engineering, genomic selection and plant breeding, pest and disease detection, precision agriculture and smart farming, soil health and nutrient management, climate resilient crop development, livestock biotechnology, challenges and ethical considerations in AI based agricultural biotechnology. Furthermore, this review addresses the exponential growth of commercial intellectual property in the field and contrast it with academic publication outputs. Finally, we critically assess the ethical challenges impeding equitable adoption of AI including data sovereignty and digital divide, while projecting future frontiers involving quantum computing. This review will help build sustainable agricultural systems capable of adapting to climate change, contribute to the development of climate-resilient and high-yielding crops, and address global food security challenges.

Agriculture↗

PubMed2026

Generation of a STRAIGHT-IN Dual AAVS1 hiPSC line with orthogonal landing pads for versatile DNA payload integration.

The STRAIGHT-IN platform is designed for facile genomic integration of DNA payloads into human induced pluripotent stem cells (hiPSCs) that contain a pre-inserted landing pad (LP). Here, we expanded the versatility of STRAIGHT-IN by introducing an additional, orthogonal LP into the unmodified allele of the safe harbor locus AAVS1. Specifically, we targeted the hiPSC line LUMC0099iCTRL04_AAVS1-bxb-v2 (hPSCreg LUMCi004-A-1), which already carried one LP. The resulting STRAIGHT-IN AAVS1 Dual line can integrate two independent DNA payloads in parallel, expanding the applicability of the platform for complex genomic engineering applications.

Humans↗

PubMed2025

Analog epigenetic memory revealed by targeted chromatin editing.

Cells store information by means of chromatin modifications that persist through cell divisions and can hold gene expression silenced over generations. However, how these modifications may maintain other gene expression states has remained unclear. This study shows that chromatin modifications can maintain a wide range of gene expression levels over time, thus uncovering analog epigenetic memory. By engineering a genomic reporter and epigenetic effectors, we tracked the gene expression dynamics following targeted perturbations to the chromatin state. We found that distinct grades of DNA methylation led to corresponding, persistent gene expression levels. Altering the DNA methylation grade, in turn, resulted in permanent loss of gene expression memory. Consistent with experiments, our chromatin modification model indicates that analog memory arises when the positive feedback between DNA methylation and repressive histone modifications is lacking. This discovery will lead to a deeper understanding of epigenetic memory and to new tools for synthetic biology.

Epigenesis, Genetic↗

PubMed2026

Targeted genomic integration and rearrangement using prime assembly.

Although therapeutic genome editing holds great potential to remedy diverse inherited and acquired disorders, targeted installation of medium-to-large genomic modifications in therapeutically relevant cells remains challenging1. Here we develop prime assembly, an approach that permits DNA sequence assembly and integration in human cells leveraging CRISPR-targeted dual flap synthesis. This method enables RNA-programmable site-specific integration of single or double-stranded DNA fragments. Unlike homology-directed repair, prime assembly is similarly active in dividing and non-dividing cells. We applied prime assembly to perform targeted exon recoding, transgene integration and megabase-scale rearrangements, including at therapeutically relevant loci in primary human cells. Prime assembly expands the capabilities of genome engineering by enabling the targeted integration of medium to large-sized DNA sequences without relying on double-stranded DNA donors, nuclease-driven double-strand breaks or cell cycle progression.

Journal Article↗

PubMed2026

Establishment of an in vitro culture and regeneration protocol for the native Chilean grass Polypogon australis Brong.

Polypogon australis Brong. is a native Chilean grass frequently found colonizing metal-rich mine tailings, yet it lacks an established in vitro regeneration system to support controlled physiological and biotechnological studies. Here, we report a reproducible protocol for seed germination, callus induction, and plant regeneration using coleoptile-mesocotyl explants. Surface-sterilized seeds were germinated on Murashige and Skoog (MS) medium supplemented with sucrose, achieving a cumulative germination percentage of 47.67 ± 3.15% after 15 days. The coleoptile-mesocotyl explant proved highly responsive to culture on callus induction medium (CIM) supplemented with dicamba, resulting in a callus induction frequency of 30.55 ± 11.96% after 3-5 weeks. Induced calli were predominantly embryogenic, with embryogenic calli representing 65.42 ± 8.61% of the total callus population. Embryogenic calli regenerated complete plantlets with a regeneration efficiency of 45.0 ± 23.3%. Organogenic structures, including primary shoots and roots, developed directly from embryogenic calli maintained on callus induction medium (CIM) supplemented with dicamba, without transfer to a specialized regeneration medium containing organogenesis-promoting growth regulators. After the initiation of organogenesis, cultures were exposed to a 16 h light/8 h dark photoperiod while remaining on CIM, and regenerated plantlets were subsequently transferred to MS+10 S medium for further growth and elongation. This study establishes the first complete in vitro regeneration system for P. australis, providing a practical framework for future physiological studies, large-scale propagation, genetic transformation, and genome engineering applications in this ecologically relevant native Chilean grass.

Regeneration↗

PubMed2024

Suppression of CNS APOE4 Expression by miRNAs Delivered by the S2 AAVrh.10 Capsid-Modified AAV Vector.

The homozygous Apolipoprotein E (APOE4) genotype is the major risk factor for the development of early Alzheimer's disease. Genome engineering studies in mouse models of human APOE4-dependent pathology have established that reduction of APOE4 expression can rescue the phenotype. We hypothesized that APOE4 could be suppressed in the CNS of APOE4 homozygotes using adeno-associated virus (AAV) expression of microRNAs (miRNA) designed to hybridize to APOE mRNA. We screened nine different miRNAs targeting APOE following transfection in HEK293T and Huh7 cells. Optimal APOE suppression was obtained with mir2A (targeting coding region nt330-351) and mirN4 (3' untranslated region nt1142-1162). miRNA expression cassettes were designed with two copies of each of these two miRNAs co-expressed with a mCherry transgene. To optimize delivery of these miRNAs, an engineered AAVrh.10 variant was identified from a screen of multiple peptide insertions into capsid loop IV and substitutions in loop VIII. This led to identifying the AAV.S2 capsid with enhanced transduction of both neurons and glia and enhanced distribution in the brain. The engineered capsid was used to deliver the APOE miRNA suppression cassette to the hippocampus of TRE4 mice (human APOE4 knock-in replacement of the murine apoE locus). Two weeks after intra-hippocampus administration, regional expression of miRNA at the injection site was quantified at the mRNA level relative to an endogenous reference. The AAV.S2 capsid provided 2.31 ± 0.37-fold higher expression of miRNA over that provided by AAVrh.10 (p < 0.05). In the targeted region, a single intra-hippocampus AAV.S2 administration suppressed hippocampal APOE4 mRNA levels by 76.5 ± 3.9% compared with 41.3 ± 3.3% with the same cassette delivered by the wildtype AAVrh.10 capsid (p < 0.0001). We conclude that an expression cassette with two different miRNAs targeting APOE4 delivered by the AAV.S2 capsid will generate highly significant suppression of APOE4 in the CNS.

Dependovirus↗

PubMed2026

Long-term rock dove (Columba livia) primordial germ cell culture: A tool towards avian conservation.

Primordial germ cells (PGCs) are critical tools for genome engineering and conservation in birds. Although culture systems for chicken PGCs have been well established for nearly two decades, efforts to propagate PGCs from other avian species have proved exceptionally challenging, limiting the broader application of artificial reproductive technologies in birds. Here we report the first successful derivation and long-term culture of PGCs from the rock dove, or common pigeon (Columba livia). Guided by transcriptomic profiling of PGCs, we developed a species-specific medium that supports PGC maintenance and expansion. We identify insulin signaling as a requirement for survival under the conditions tested, and demonstrate that inhibition of the retinoic acid receptor, in the presence of vitamin A, supports propagation. Supplementation with bone morphogenetic protein 4, leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor, and pleiotrophin further enhances PGC proliferation. Cultured cells express canonical germline markers and migrate to the gonads following injection into both rock dove and chicken embryos, confirming functional migratory competency. These findings establish a platform for germline manipulation and biobanking in Columbidae, broadening the potential applicability of reproductive technologies to conservation efforts.

Animals↗

PubMed2026

Enhanced exonuclease-Cas9 systems promote multiple nucleotide deletions with higher efficiency and broader targeting scope in plants.

CRISPR-Cas9 is a widely used platform for plant genome editing, but its outcomes are typically dominated by small insertions and deletions (indels). Such limited mutation profiles restrict its utility in functional studies of non-coding RNAs and regulatory elements, such as microRNAs (miRNAs), untranslated regions (UTRs), and promoter sequences, where larger sequence disruptions are often required. Here, we developed enhanced exonuclease-Cas9 platforms, termed multiple nucleotide deletion Cas9 (MND-Cas9) systems, for efficient generation of large deletions in rice. By screening four exonucleases (RecJ, T5, TREX2, and SbcB), we established MND-Cas9v1 systems based on TREX2 or SbcB that produced substantially larger deletions without reducing editing efficiency. Further optimization with an inserted DNA-binding domain (DBD) between Cas9 and exonuclease yielded MND-Cas9v2, which simultaneously enhanced efficiency and deletion size. To expand PAM compatibility, we introduced PAM-relaxed Cas9-NG and SpG variants, generating MND-Cas9-NG/SpGv2 systems with broader targeting scope and superior performance compared to their parental nucleases. Finally, we demonstrated the utility of these systems in two applications: MND-Cas9v2 efficiently knocked out the miRNA gene OsMIR530, producing larger seeds, and generated extended deletions in the 3'UTR of OsGhd2, which upregulated its expression and increased grain size. These results demonstrate that MND-Cas9 systems enable high-efficiency generation of extended deletions and facilitate functional analyses of non-coding RNAs and regulatory sequences. Overall, this work establishes a versatile and expandable exonuclease-Cas9 platform that substantially broadens the mutational spectrum and application potential of CRISPR-Cas9 for plant genome engineering.

CRISPR-Cas Systems↗

PubMed2025

Disassembly activates Retron-Septu for antiphage defense.

Retrons are antiphage defense systems that produce multicopy single-stranded DNA (msDNA) and hold promise for genome engineering. However, the mechanisms of defense remain unclear. The Retron-Septu system integrates retron and Septu antiphage defenses. Cryo-electron microscopy structures reveal asymmetric nucleoprotein complexes comprising a reverse transcriptase, msDNA (a hybrid of msdDNA and msrRNA), and two PtuAB copies. msdDNA and msrRNA are essential for assembling this complex, with msrRNA adopting a conserved lariat-like structure that regulates reverse transcription. Notably, the assembled Retron-Septu complex is inactive, with msdDNA occupying the PtuA DNA binding site. Activation occurs upon disassembly, releasing PtuAB, which degrades single-stranded DNA to restrict phage replication. This "arrest-and-release" mechanism underscores the dynamic regulatory roles of msDNA, advancing our understanding of antiphage defense strategies.

Cryoelectron Microscopy↗

PubMed2026

OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides.

Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g., over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually ≤ 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information, such as binary (digital) data in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides - particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis - remain inadequate. To address this gap, OligoSeq is presented as an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation with sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than ≥ 90% identity spanning from about 70% to 10% with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method. OligoSeq can be integrated with nanopore sequencing technology pipelines and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC/MS.

Sequence Analysis, DNA↗

PubMed2026

When Homing Endonuclease Meets Transposon: The OMEGA System.

Sequence-specific DNA endonucleases have made significant contributions to biology, biotechnology, and medicine; restriction enzymes and homing endonucleases are among classic examples. The demonstration of programmable genome editing using Cas9 in the CRISPR-Cas system, in which the target DNA sequence is recognized by base pairing with a guide RNA, revolutionized the field of genome engineering, making target selection more flexible and convenient. The OMEGA (Obligate Mobile Element-Guided Activity) system, considered a precursor to Cas12, and likely to Cas9, in the CRISPR-Cas system, is an RNA-guided DNA endonuclease composed of a TnpB, IscB, IsrB, or Fanzor protein, and a structural RNA designated reRNA or ωRNA. The OMEGA system is present in the three domains of life as an auxiliary component of transposons. The OMEGA system cuts DNA in an allele from which a transposon is excised and triggers recombination to reinstate the transposon at the same position. This "transposon restorative homing" redefines the OMEGA system as a homing endonuclease. In this review, the selfish aspects of the OMEGA system are discussed in the historical context of homing endonuclease research.

Cas12↗

PubMed2023

Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.

Escherichia coli↗

PubMed2026

Distinct YY dinucleotide periodicity in adeno-associated virus DNA.

Dinucleotide periodicity is a hallmark of genome organization, yet its role in single-stranded (ss)DNA viruses remains poorly understood. Here, we systematically analyzed dinucleotide spacing patterns in adeno-associated virus (AAV) genomes and other viruses. Across 13 primate AAV serotypes, we identified a pronounced and highly conserved ∼15-bp periodicity specific to pyrimidine-pyrimidine (YY) dinucleotides and their reverse complements (RR). Comparative analyses across >25,000 viral sequences demonstrate that this 15-bp YY/RR periodicity is unique to the genus Dependoparvovirus and absent from other ssDNA viruses, satellite viruses, and helper viruses, which predominantly exhibit canonical ∼10- to 11-bp periodicities. Upon disruption of the YY/RR pattern using DNA family shuffling of AAV capsid genes, and subsequent iterative selection for viral production or cell entry, we found that the pattern is under positive selection. Selected sequences display increased periodicity alongside reduced sequence diversity, supporting a functional role for this genomic feature. Finally, engineered recombinant AAV genomes containing YY periodic motifs exhibit enhanced production and, for some designs, improved transduction efficiency, demonstrating that YY periodicity can modulate viral replication and infectivity. Our findings uncover a unique DNA-encoded signal in dependoparvoviruses that contributes to AAV fitness, expands our knowledge of virus biology, and has implications for vector engineering.

Dependovirus↗

PubMed2026

Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding.

This review synthesizes the molecular mechanisms of cold tolerance in pepper, integrating multi-omics data,genome editing, and precision breeding strategies to accelerate the development of cold-resilient cultivars. Cold stress is a significant environmental factor that affects the growth, productivity, and fruit quality of Capsicum annuum by impairing membrane integrity photosynthesis and cellular redox homeostasis. Although pepper has several endogenous cold-responsive regulators such as CaNAC035 and CabHLH035, along with antioxidant defense systems, its cold tolerance remains limited due to low transcriptional activation of key regulators, functional redundancy among cold-responsive genes, and the polygenicity of cold tolerance. These complexities, combined with low genetic diversity and linkage drag, have hindered the improvement of cold-resistant cultivars through conventional breeding. This review brings together the recent progress in understanding the molecular mechanisms of cold stress perception, signal transduction, transcriptional regulation, metabolic reprogramming, and phytohormone interactions in pepper. Precision Breeding 2.0 is a new innovation that combines the integration of multi-omics-based target identification with next-generation genome-editing techniques, allowing precise and multiplex engineering of complex and interconnected regulatory networks instead of single genes. We cover new approaches such as engineering the DREB/CBF pathway, allele-specific editing and targeted disruption of negative regulators to enhance the pathway(s) involved in cold response. Moreover, we propose a roadmap for integration of transcriptomics, proteomics, metabolomics, high-throughput phenomics, and speed breeding to accelerate the identification, validation, and deployment of superior alleles to boost cold tolerance. This review provides a foundation for developing climate-resilient pepper cultivars by connecting functional genomics with precision genome engineering approaches to maintain productivity under variable environmental conditions.

Capsicum↗

PubMed2025

Quantifying Protein-Nucleic Acid Interactions for Engineering Useful CRISPR-Cas9 Genome-Editing Variants.

Numerous high-specificity Cas9 variants have been engineered for precision genome editing. These variants typically harbor multiple mutations designed to alter the Cas9-single guide RNA (sgRNA)-DNA complex interactions for reduced off-target cleavage. By dissecting the contributions of individual mutations, we attempt to derive principles for designing high-specificity Cas9 variants. Here, we computationally modeled the specificity harnessing mutations of the widely used Cas9 isolated from Streptococcus pyogenes (SpCas9) and investigated their individual mutational effects. We quantified the mutational effects in terms of energy and contact changes by comparing the wild-type and mutant structures. We found that these mutations disrupt the protein-protein or protein-DNA contacts within the Cas9-sgRNA-DNA complex. We also identified additional impacted amino acid sites via energy changes that constitute the structural microenvironment encompassing the focal mutation, giving insights into how the mutations contribute to the high-specificity phenotype of SpCas9. Our method outlines a strategy to evaluate mutational effects that can facilitate rational design for Cas9 optimization.

Gene Editing↗

PubMed2026

De-extinction technology and its application to conservation.

De-extinction, once the realm of science fiction, has evolved into a tangible scientific endeavor thanks to breakthroughs in genome sequencing, engineering, advanced assisted reproductive technologies, and stem cell biology. Alongside this work are innovations in reintroduction science and artificial intelligence, which are refining strategies for species translocations, rewilding, and long-term ecosystem monitoring of de-extinct species and populations. While the primary motivation for de-extinction is restoring lost ecological functions to eroded ecosystems, each of these technologies can also be applied to conservation biology for de-endangerment, offering new solutions for biodiversity preservation. This review synthesizes the technological advancements emerging from de-extinction science and explores their broad applications in conservation, demonstrating how de-extinction is both about resurrecting lost species and about expanding the conservation toolkit to sustain and rebuild biodiversity in the face of accelerating environmental change.

Conservation of Natural Resources↗

PubMed2023

Water a major source of endocrine-disrupting chemicals: An overview on the occurrence, implications on human health and bioremediation strategies.

Endocrine disrupting chemicals (EDCs) are toxic compounds that occur naturally or are the output of anthropogenic activities that negatively impact both humans and wildlife. A number of diseases are associated with these disruptors, including reproductive disorders, cardiovascular disorders, kidney disease, neurological disorders, autoimmune disorders, and cancer. Due to their integral role in pharmaceuticals and cosmetics, packaging companies, agro-industries, pesticides, and plasticizers, the scientific awareness on natural and artificial EDCs are increasing. As these xenobiotic compounds tend to bioaccumulate in body tissues and may also persist longer in the environment, the concentrations of these organic compounds may increase far from their original point of concentrations. Water remains as the major sources of how humans and animals are exposed to EDCs. However, these toxic compounds cannot be completely biodegraded nor bioremediated from the aqueous medium with conventional treatment strategies thereby requiring much more efficient strategies to combat EDC contamination. Recently, genetically engineered microorganism, genome editing, and the knowledge of protein and metabolic engineering has revolutionized the field of bioremediation thereby helping to breakdown EDCs effectively. This review shed lights on understanding the importance of aquatic mediums as a source of EDCs exposure. Furthermore, the review sheds light on the consequences of these EDCs on human health as well as highlights the importance of different remediation and bioremediation approaches. Particular attention is paid to the recent trends and perspectives in order to attain sustainable approaches to the bioremediation of EDCs. Additionally, rigorous restrictions to preclude the discharge of estrogenic chemicals into the environment should be followed in efforts to combat EDC pollution.

Animals↗