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PubMed2026

CRISPRessoSea: streamlined analysis and comparison of pooled amplicon CRISPR screens.

BACKGROUND: CRISPR genome editing enables precise modification of genomic targets but may also induce unintended edits at off-target sites with similar sequences. Pooled amplicon sequencing can assess on- and off-target editing across many samples, yet analyzing, aggregating, and visualizing results from multiple pooled experiments remains challenging. Tools to simplify and standardize these analyses are needed to provide reproducible and comparable interpretation of editing data. RESULTS: We developed CRISPRessoSea, a software package that processes, compares, and visualizes genome editing rates from pooled amplicon sequencing experiments. The tool provides standardized workflows for analyzing editing across multiple targets and samples, supports both nuclease- and base-editing modalities, and generates clear, data-rich summaries suitable for downstream interpretation. CONCLUSIONS: CRISPRessoSea facilitates reproducible, scalable analysis of CRISPR editing outcomes across diverse experimental designs, enabling more efficient and transparent assessment of genome editing specificity. The software is freely available at https://github.com/clementlab/CRISPRessoSea .

Software↗
PubMed2026

Loss of Ku70 promotes mononucleate conidiation and homologous recombination in Phanerochaete chrysosporium.

Lignin is a major constituent of lignocellulose and the most abundant aromatic biopolymer on earth. It provides plants with rigidity and protection, but its recalcitrant nature also presents a significant barrier to lignocellulose valorization. The white-rot fungus Phanerochaete chrysosporium is among nature's most efficient lignin degraders, and its ligninolytic capabilities have been subjected to intensive investigations. Genome editing with precision is crucial for elucidating the in vivo mechanisms of its ligninolytic actions, but genetic manipulations of P. chrysosporium are often plagued by imprecision. This technical nuisance is driven primarily by canonical non-homologous end joining (c-NHEJ), a DNA repair system that requires little homology and depends on the binding of the Ku70/Ku80 heterodimer to double-strand break (DSB) ends. Loss of Ku70 or Ku80 abolishes c-NHEJ and significantly improves genome editing precision in many filamentous fungi, but it has yet to be examined and exploited in P. chrysosporium. Here, we constructed a homozygous ku70Δ mutant in a meiotic homokaryon of clear genetic background. Loss of Ku70 minimally impacts growth but significantly increases homologous recombination frequency from ~2% to ~66%, with ~32% of the latter being homozygous. Unexpectedly, loss of Ku70 also promotes mononucleate conidiation, which may facilitate isolation of homozygous mutants. Taken together, our work provides a valuable genetic tool to understand and exploit P. chrysosporium's remarkable ligninolytic capabilities.IMPORTANCEGenome editing with precision is essential to unraveling the intricacies of P. chrysosporium's exceptional ligninolytic capabilities, but the available tools are generally imprecise due to the dominance of non-homologous recombination, a problem that is further exacerbated by the discontinuation of Novozyme 234. We tackle these challenges by reestablishing protoplast-based transformation with Lywallzyme as an alternative. Importantly, we demonstrate that inactivation of c-NHEJ by deleting ku70 significantly increases gene knockout efficiency and report the unexpected involvement of c-NHEJ in regulating the number of nuclei during conidiation. Our work paves the way for future ventures into understanding ligninolysis in P. chrysosporium and building superior chassis for industrial applications.

Ku70↗
PubMed2026

From scissors to editors: how the evolution of precision is redefining therapeutic genome editing.

Since its introduction as a genome-editing tool, CRISPR-based technology has undergone rapid refinement, with precision emerging as a central focus of development. Early CRISPR-Cas9 systems demonstrated unprecedented ease and efficiency in targeting specific DNA sequences, but concerns over off-target effects and variable editing outcomes limited their broader application. This review outlines the progression of CRISPR from its discovery in prokaryotes to its application as a versatile tool in precision medicine, where it supports targeted therapies for genetic disorders in various ways. Although technical challenges, including off-target editing and delivery inefficiencies, persist alongside ethical considerations of accessibility and long-term consequences, CRISPR's ongoing refinements and innovations reflect a clear trajectory toward greater specificity, safety, and predictability, positioning CRISPR as an increasingly precise platform for both fundamental research and therapeutic use.

Gene Editing↗
PubMed2025

Bacterial directed evolution of CRISPR base editors.

Base editing and other precision editing agents have transformed the utility and therapeutic potential of CRISPR-based genome editing. While some native enzymes edit efficiently with their nature-derived function, many enzymes require rational engineering or directed evolution to enhance the compatibility with mammalian cell genome editing. While many methods of engineering and directed evolution exist, plate-based discrete evolution offers an ideal balance between ease of use and engineering power. Here, we describe a detailed method for the bacterial directed evolution of CRISPR base editors that compounds technical ease with flexibility of application.

Gene Editing↗
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↗
PubMed2026

Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing.

CRISPR-based prime editors (PEs) install precise edits into genomic DNA without generating double-strand breaks. Their editing efficiency is highly dependent on reverse transcriptases (RTs), but efficient RT candidates remain limited. Here, we identified 19 novel active RTs by screening 558 candidates. Among them, RERV-RT, derived from Rattus norvegicus, exhibited the highest activity. Through structure-guided engineering and deep mutational scanning, we developed an optimized variant, enRERV-RT, which outperforms conventional M-MLV-RT-based PE systems by 1.20-fold in mammalian and plant cells, and by 1.88-fold at hard-to-edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high-throughput platform, TRAP-seq-PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV-RT exhibited higher editing efficiencies than those based on M-MLV-RT. Collectively, our work establishes a versatile, high-efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.

Animals↗
PubMed2026

Advances in Precision Editing Therapies for Alpha-1 Antitrypsin Deficiency.

Genome and RNA editing modalities have revolutionized precision gene therapy, offering a safer alternative to traditional gene replacement approaches. Alpha-1 antitrypsin deficiency (AATD) is a compelling model for precision medicine because the disease mechanism is well defined-mutations in a single gene are responsible for both liver and lung pathology. In this review, we summarize the current preclinical and clinical efforts for AATD, with an emphasis on genome and RNA editing strategies.

Humans↗
PubMed2026

Leaf Rust in Rye: From Pathogen Biology to Host Defense and Resistance Breeding.

Leaf rust (LR), caused by Puccinia recondita f. sp. secalis (Prs), is considered one of the most dangerous rye (Secale cereale L.) diseases, causing yield losses exceeding 35%. This review summarizes all currently available data about this disease: pathogen characteristics (including its life cycle, natural variation, and disease symptoms), resistance resources, and the background of the plant immune response at the genome, transcriptome, and metabolome levels. The research conducted so far has allowed for the identification of dozens of genes that play a significant role in the rye immune response to Prs infection. Among them, genes encoding NBS-LRR proteins (including SECCE1Rv1G0014220, the most likely Pr3 candidate), glycosyltransferase, β-1,3-glucanase, 1-deoxy-D-xylulose 5-phosphate synthase, β-1,3-glucanase, UDP-glycosyltransferase, pathogenesis-related protein 1, ammonium transporter, and cytochrome P450 enzymes are candidates for seedling and all-stage resistance, whereas ScLr_ABC25 currently represents the most promising candidate associated with adult-plant resistance. Among the metabolites differentially accumulated in response to Prs, those related to phenylpropanoids, diterpenoids, and thiamine branches seem to play the most important role in the immune response. Finally, we suggest how the knowledge acquired so far about the rye-Prs interaction can be used in modern breeding programs aimed at obtaining cultivars with enhanced resistance to LR, such as through the use of functional gene markers and/or metabolic biomarker-assisted selection and, in the more distant future, by developing and applying new genomic techniques for precise editing of resistance and susceptibility genes, engineering synthetic immune receptors and decoys, and pan-genomic exploration for identification of rare or lineage-specific resistance alleles. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plant Diseases↗
PubMed2026

From feasibility to predictability: prime editing redefines precision breeding in plants.

Originally developed in mammalian systems as a genome editing strategy without double-strand breaks, prime editing (PE) has been adapted for precise genome modifications. However, its deployment revealed key limitations, including reduced efficiency, strong locus dependency, low germline transmission, and somatic chimerism. Consequently, diverse PE variants have emerged, resulting in fragmented landscape of architectures with context-dependent and inconsistent performance. This review consolidates these advances and outlines emerging design principles behind plant PE systems. It evaluates optimization strategies at multiple levels, discusses their applications in monocots and eudicots, and highlights persistent bottlenecks and future directions, including AI-guided protein engineering and improved delivery strategies. These advances position PE as a rapidly evolving platform toward enabling precision breeding in plants.

cis-regulatory engineering↗
PubMed2026

Genome Editing in Solanaceae: Harnessing CRISPR-Cas Technology for Precision Crop Improvement.

Malnutrition and climate-induced stress remain major constraints to global food and nutritional security despite the yield gains of the Green Revolution. Solanaceae crops such as tomato, potato, brinjal, and pepper are key sources of vitamins, minerals, and bioactive compounds. Yet, their genetic improvement has been limited by narrow diversity and complex polygenic traits. The advent of CRISPR/Cas-mediated genome editing provides a transformative platform for precision crop improvement by enabling targeted modification of genes controlling stress tolerance, yield, and nutritional quality. In Solanaceae, CRISPR/Cas applications have successfully enhanced resistance against major pathogens (SlMlo1, SlPelo, SlDCL2), improved abiotic stress tolerance through editing of SlMAPK3, SlCBF1, and SlBZR1, and optimized fruit quality traits via modulation of Psy1, CrtR-b2, and fiAD2/3. Emerging innovations, such as base and prime editing, and RNP-mediated transgene-free delivery, are expanding the precision and scope of editing. However, challenges persist, including genotype-dependent transformation, low HDR efficiency, and incomplete understanding of off-target and epigenetic effects. Integrating CRISPR with omics-guided gene discovery, efficient transformation systems, and regulatory harmonization can accelerate the development of nutritionally enriched, stress-resilient, and sustainable Solanaceae varieties. This review synthesizes recent advances, identifies critical limitations, and outlines future opportunities for deploying CRISPR/Cas technology to achieve next-generation breeding and food system resilience.

CRISPR/Cas↗
PubMed2025

Efficient CRISPR/Cas9-mediated genome editing of phytoene desaturase in Musa-AAA: a critical step for genetic improvement of east African highland bananas.

East African highland bananas (EAHBs), locally referred to as "matooke", are an important staple crop in Uganda. The EAHBs have a triploid genome (AAA) with a large phenotypic diversity in the Great Lakes region of Africa and are challenged by both abiotic and biotic factors. The EAHBs have been improved through conventional breeding and genetic engineering though facing challenges such as genetic drag of unfavorable traits and complex regulatory processes, respectively. Therefore, a more precise approach for crop improvement such as genome editing is highly recommended. In the current study, we assessed the feasibility and applicability of the CRISPR/Cas9 mediated-genome editing in EAHBs. Two sgRNAs were designed from the Nakitembe phytoene desaturase (PDS) gene and used to edit the PDS gene in Nakitembe (NKT) and NAROBan5 (M30) cultivars. A total of 47 NKT and 130 M30 events were regenerated via agrobacterium-mediated transformation of banana embryogenic cell suspensions. Up to 100% and 94.6% albinism rates were observed in Nakitembe and M30 cultivars respectively with additional albino-variegated and variegated phenotypes observed in M30 only. Carotenoid analysis revealed a significant reduction of total carotenoid content in edited events with all complete albinos showing no detectable carotenoids implying that the carotenoid biosynthetic pathway was effectively disrupted. Sequence analysis revealed that all of the edited events had frameshift mutations leading to PDS disruption. Overall, this study presents the first report of CRISPR/Cas9 genome editing in EAHBs and more interestingly on a hybrid, M30 showing high precision and efficiency. This validated genome editing system provides a robust platform for targeted EAHB improvement.

CRISPR/Cas9↗
PubMed2026

Efficient prime editing in vivo and in vitro using lipid nanoparticles.

Prime editing is a versatile clinical genome editing method that enables precise substitutions, small insertions and deletions at specified locations in the genomes of living systems including human cells. Although non-viral lipid nanoparticle (LNP) delivery of RNA in vivo has become a preferred method for gene editing in animals and patients, its application to complex, three-component prime editing systems has yielded low editing efficiencies. Here we developed a systematic prime editing LNP (PE-LNP) optimization platform that addresses key bottlenecks in cargo design that limit editing efficiency. This generalizable workflow yielded PE-LNPs that can achieve 49% average in vivo prime editing in the bulk mouse liver with a single dose of 2 mg kg-1. We applied our workflow to the correction of PAH R408W, a cause of phenylketonuria, in a mouse model and achieved prime editing efficiencies and serum phenylalanine levels anticipated to be curative. We also show that PE-LNPs minimize off-target editing compared with DNA delivery methods, induce only transient elevation of liver enzymes and can be dosed repeatedly to improve editing efficiencies. These PE-LNP systems provide an attractive alternative to viral delivery by offering transient expression that minimizes off-target editing, no observed long-term toxicity and high levels of non-viral in vivo liver prime editing.

Animals↗
PubMed2026

Herbicide Resistance Genes in Crops: Mechanisms, Progress, and Future Perspectives.

While previous reviews have largely focused on individual crops or single target-site mechanisms, the full-chain comparative landscape across major cereal crops remains unexplored. Here, we fill this critical gap by providing the first systematic, cross-crop comparative review that spans herbicide targets, resistance mechanisms, and breeding applications across four major cereals-rice, maize, wheat, and sorghum. Weed infestation is a serious constraint on crop production. Chemical weed control faces challenges such as herbicide resistance evolution and ecological risks. Developing herbicide-resistant varieties is a fundamental approach to achieve green and sustainable weed management. This review systematically summarizes research progress on herbicide resistance genes from three aspects: herbicide classification, resistance mechanisms, and crop breeding applications. It highlights key differences among four major cereal crops (rice, maize, wheat, and sorghum) in resistance-gene discovery and translational progress. Rice has the richest target-site resistance-gene resources. Maize leads in commercialization of transgenic herbicide resistance. Wheat focuses on endogenous precise editing due to genome complexity and regulatory constraints. Sorghum relies on specific mutations to serve cereal-legume intercropping systems. Based on this comparison, this review identifies the core trends in resistance breeding: from single-gene to multi-gene stacking, and from exogenous gene introduction to endogenous gene editing. It also points out common bottlenecks, including insufficient systematic mining of resistance-gene resources, lagging elucidation of non-target-site resistance regulatory networks, and strong genotype dependence in genetic transformation. Future efforts should focus on exploring broad-spectrum resistance genes, optimizing precise editing technologies, and developing sustainable resistance management strategies. This review provides a theoretical framework and practical references for molecular breeding of herbicide-resistant crops.

crop breeding↗
PubMed2026

Synergistic HMGN1 and VP64 Fusions Potentiate High-Precision and PAM-Flexible Base Editing.

RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.

Gene Editing↗
PubMed2026

ALPINE: a scalable pipeline for comprehensive classification of gene-editing outcomes from long-read amplicon sequencing.

SUMMARY: CRISPR genome editing has enabled precise genetic modification for gene and cell therapies, but edits often produce heterogeneous on-target outcomes, including homology-directed repair (HDR) knock-ins, DNA repair template integrations, and structural variants. Existing tools are frequently limited to short reads or lack viral vector-specific integration categories needed for therapeutic development. Here, we present ALPINE (Amplicon Long-read Pipeline for INtegration Evaluation), a scalable and reproducible pipeline for classifying and quantifying gene-editing outcomes from long-read amplicon sequencing supporting both PacBio HiFi and Oxford Nanopore platforms. ALPINE classifies reads into 10+ categories, including DNA repair vector integration subtypes, and performs variant calling near the gene-edited site with batch, multi-sample reporting. Uniquely, ALPINE can distinguish between cells treated with multiple DNA repair vectors and identify distinct molecular features, such as inverted terminal repeats (ITRs), enabling comprehensive characterization of complex gene editing outcomes. Dual-target benchmarking on simulated datasets demonstrated high accuracy for transgene integration events. Independent validation on public crosslinked-HDR dataset confirmed ALPINE's integration detection capabilities, and application to edited T cell samples demonstrated comprehensive gene-editing outcome profiling. AVAILABILITY: ALPINE is available under MIT license at https://github.com/Maggi-Chen/ALPINE and https://doi.org/10.5281/zenodo.20272510. All analysis scripts and visualization code used in this manuscript are available at https://github.com/Maggi-Chen/ALPINE-manuscript-analysis. Simulated datasets are deposited at Zenodo (https://doi.org/10.5281/zenodo.20260865). Public dataset PRJNA913199 is available through NCBI SRA.

Gene Editing↗
PubMed2026

Powering Genome Editing in Rice by Harnessing Promising Gene Resources: A Comprehensive Roadmap.

The imprecise breeding methods including recombination breeding, physical/chemical mutagenesis, and marker-assisted breeding have been extensively utilized for trait improvement of rice crop. Despite tremendous progress made through these breeding methods, the critical issues, such as linkage drag, unintended phenotype, and longer duration of time required to breed a cultivar, have been the major limitations. Among the new breeding technologies, genome editing (GE) has become the most promising approach because of its specificity, precision, and speed. Despite its transformative potential, genome editing continues to face several limitations in crop improvement. These include well-recognized policy challenges, such as biosafety regulations and intellectual property constraints, alongside technical barriers like inefficient tissue culture and transformation systems. Additionally, researchers remain constrained by the limited availability of precise gene information necessary for accurate targeted editing and effective trait enhancement. This review presents an analysis of genes that regulate abiotic and biotic stresses, yield, grain quality and nutrition, plant architecture, nutrient absorption and use efficiency, and other agronomically important traits of rice. The trait-wise probable target genes for genome editing have been discussed in detail. This review will serve as a ready reckoner for rice researchers and funding agencies.

Oryza↗
PubMed2026

Emerging trends in genome editing of wild animals.

Globally, nearly one million species are currently threatened with extinction, highlighting the need for more efficient solutions to biological conservation. Genome editing, which allows for faster and more precise changes in genomes, is a promising technique for boosting populations through facilitated adaptation, management of invasive or pathogenic populations, and potentially even facilitating the revival of extinct species. These approaches belong to a new field of research termed conservation biotechnology, which places a great responsibility on researchers and decision makers to ensure sustainability. In this paper, we have mapped the emerging trends in genome editing of wild animals. Current projects primarily focus on population control and de-extinction, with fewer initiatives aimed at preserving threatened species. We then explore four critical dimensions of conservation biotechnology: the technology itself, new perspectives on conservation practices, research organization, and governance and policy. Despite its potential, key questions remain-particularly whether genome editing can increase genetic diversity without causing unintended non-target impacts. Genome editing also provokes new perspectives on conservation practices where ecosystem-wide impact assessment, case-by-case evaluations, and post-release monitoring needs to be prioritized. Furthermore, conservation biotechnology is heavily funded through private funding showing varying stakeholder interest, which can lead to untraditional and less transparent research processes. Stakeholders, including local and indigenous people, are only to a certain degree involved, which may weaken inclusion of local knowledge and monitoring efforts. Finally, concerning governance and policy, there is an urgent need to develop more adequate regulation of conservation biotechnology, as environmental release of genome-edited animals challenges definitions and guidelines in current nature protection laws and GMO regulations. Based on our analysis, we outline key points for further investigation toward a more sustainable approach to conservation biotechnology.

Animals↗