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PubMed2026

Validation of the TransplantTrace cfDNA Kidney assay for measurement of donor-derived cell-free DNA in transplant recipients.

INTRODUCTION: Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising non-invasive marker for assessing allograft status and guiding clinical management in transplant recipients. Its utility in kidney transplantation has repeatedly been demonstrated in large studies showing a strong association between elevated dd-cfDNA levels and allograft injury or rejection. This study evaluated the performance of a centralized next-generation sequencing (NGS)-based assay for measurement of dd-cfDNA in patients post-kidney transplantation. METHODS: The TransplantTrace cfDNA Kidney assay utilizes 50 insertion-deletion (indel) markers to discriminate dd-cfDNA. Evaluation of analytical performance included determination of input requirements, analytical sensitivity and specificity, as well as accuracy and precision parameters. Diagnostic performance was evaluated in a retrospective cohort of 104 post-transplantation samples by comparing dd-cfDNA results with biopsy-confirmed rejection. RESULTS: The assay required low DNA input (2 ng) and demonstrated high analytical sensitivity, with a verified limit of detection of 0.2% and limit of quantification of 0.3% dd-cfDNA. Analytical accuracy was excellent (R 2 = 1.00), with high repeatability and reproducibility across the reportable range of 0.2-30% dd-cfDNA. In the clinical validation, the assay showed high concordance with biopsy-confirmed rejection and excellent discriminatory performance for differentiating active from non-active rejection (AUC of 0.980). At a 1% cut-off, the assay exhibited a positive predictive value of 100%, supporting confident identification of patients likely to have treatable graft injury, while a high negative predictive value (95.6% at 15% prevalence) supports its reliability in ruling out active rejection. CONCLUSION: The TransplantTrace cfDNA Kidney assay demonstrated robust analytical performance and strong clinical concordance with biopsy-confirmed rejection status. Its high diagnostic accuracy supports reliable identification and exclusion of active rejection, with the potential to reduce reliance on invasive biopsy procedures in patients with elevated serum creatinine but low dd-cfDNA levels. In summary, the findings of this study support the implementation and use of this centralized assay for measurement of dd-cfDNA in patients post-kidney transplantation.

centralized↗
PubMed2025

Algorithms to reconstruct past indels: The deletion-only parsimony problem.

Ancestral sequence reconstruction is an important task in bioinformatics, with applications ranging from protein engineering to the study of genome evolution. When sequences can only undergo substitutions, optimal reconstructions can be efficiently computed using well-known algorithms. However, accounting for indels in ancestral reconstructions is much harder. First, for biologically-relevant problem formulations, no polynomial-time exact algorithms are available. Second, multiple reconstructions are often equally parsimonious or likely, making it crucial to correctly display uncertainty in the results. Here, we consider a parsimony approach where only deletions are allowed, while addressing the aforementioned limitations. First, we describe an exact algorithm to obtain all the optimal solutions. The algorithm runs in polynomial time if only one solution is sought. Second, we show that all possible optimal reconstructions for a fixed node can be represented using a graph computable in polynomial time. While previous studies have proposed graph-based representations of ancestral reconstructions, this result is the first to offer a solid mathematical justification for this approach. Finally we provide arguments for the relevance of the deletion-only case for the general case.

Algorithms↗
PubMed2026

Genome-Wide Identification of SSR and InDel Markers and Experimental Validation of SSR Markers for Distinguishing Cold-Tolerant and Cold-Sensitive Lily Cultivars.

In this study, whole-genome resequencing was performed on the cold-tolerant variety ND-6 and the cold-sensitive variety 'Sorbonne'. After evaluation, the Lilium davidii var. unicolor reference genome was selected to analyze SSR distribution characteristics. Whole-genome InDel identification and comparative analysis were conducted for the two varieties, yielding 34,812,909 and 24,497,857 InDels, respectively. Short InDels were predominant, with deletions slightly outnumbering insertions, mostly located in intergenic regions. Twenty pairs of SSR primers were screened and synthesized. Among them, 10 pairs amplified clearly, with a polymorphism rate of 82.6%, effectively distinguishing the two cultivars examined in this study. This study provides systematic data and a reliable marker resource for the analysis of lily genomic variation, laying a foundation for the identification of cold-tolerant germplasm; validation across additional cultivars and individuals will be required to extend their utility to broader germplasm.

cold resistant lilies↗
PubMed2026

Efficient and precise programmable DNA knock-in without double-strand breaks.

Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging1,2. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7 kb to more than 10 kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor 2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-T cell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-T cells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.

Animals↗
PubMed2026

Screening for dual sgRNAs with comparable indel efficiencies enhances CRISPR-mediated large-fragment deletion.

CRISPR-mediated large-fragment deletion provides a powerful approach for gene clusters, noncoding regions and structural variants, but its broader application is limited by low and variable deletion efficiency. Here, we systematically designed and evaluated 78 sgRNAs targeting nine representative gene clusters (ttn.1-ttn.2 cluster, 7 hox clusters and nppb-nppa cluster), containing 31 large fragments (5 kb-340 kb) to investigate the determinants of deletion efficiency. We found two key rules for achieving high deletion efficiency: (i) using dual sgRNAs with similar indel efficiencies, and (ii) applying a single sgRNA pair rather than multiple sgRNAs. Based on those rules, a 340 kb deletion is detected in the progenies of 95% of founders. Whereas the deletion size showed no significant linear correlation with deletion efficiency within the tested range. Implementing these rules resulted in an average of 70% of founders transmitting deletions across all tested sgRNA pairs. Therefore, screening sgRNAs can effectively enhance CRISPR utility in deletions, thereby facilitating the application of genomic manipulation in vertebrates and other species.

CRISPR↗
PubMed2025

A quick guide to evaluating prime editing efficiency in mammalian cells.

According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.

Gene Editing↗
PubMed2026

Co-occurrence of bronchiolar adenoma and lung adenocarcinoma: a study of nine cases revealing distinct clonal origins via integrated histologic, immunophenotypic and molecular analysis.

PURPOSE: This study sought to elucidate the possible biological association between BA and lung adenocarcinoma through an analysis of cases in which both lesions coexist within the same specimen. METHODS: In our cohort, the BA and lung cancer components of nine concurrent-type BAs were microdissected using the Millisect system and subjected to whole-exome sequencing (WES). Their histopathological, immunohistochemical, and genomic profiles were comparatively evaluated. RESULTS: Histopathologically, the BA regions of concurrent-type BAs exhibited a classic bilayered architecture, composed of continuous luminal and basal cell layers. The adjacent monolayered concurrent components were diagnosed as adenocarcinoma in situ (AIS, N = 4), minimally invasive adenocarcinoma (MIA, N = 2), and invasive adenocarcinoma (ADC, N = 3). Immunohistochemically, both luminal and basal cells in BA regions expressed thyroid transcription factor 1 (TTF1), albeit with more heterogeneous staining intensity compared to that observed in tumor components. Molecularly, EGFR mutations were the most frequently identified in either BA or tumor components, or in both (Case 9). In BA components, mutations included exon 19 p.S752F, exon 19 deletions (p.L747_T751delinsP and p.E746_T751delinsVP), and compound G719C/S768I mutations. Tumor components harbored exon 28 S1130C, exon 19 indel (p.E746_S752delins), and exon 18 p.G719C mutations. Notably, only three cases demonstrated limited overlap of mutations and copy number variations (CNVs) between the two components. Phylogenetic analysis revealed that six cases shared truncal alterations in genes including KMT2A, PIK3CA, SETD2, MITF, PBRM1, and SRSF3, one case harbored a shared canonical EGFR mutation (p.G719C), with an additional p.S768I alteration uniquely detected in the BA component. CONCLUSION: There is insufficient evidence to support BA as a premalignant lesion for lung adenocarcinoma base on morphological and molecular variables, and they may represent distinct pathological entities.

Concurrent-type bronchiolar adenoma↗
PubMed2026

Molecular dissection and functional characterization of the liguleless1 gene for manipulation of leaf angle in maize.

Recessive liguleless1 (lg1) gene significantly reduces leaf angle in maize and has become the choice in breeding for high plant density. Here, we sequenced the entire lg1 gene (5560 bp) among seven wild-type (Lg1) and one mutant (lg1) inbreds. The analysis revealed a total of 229 SNPs and 155 InDels within the Lg1 gene. The study also revealed the existence of three exons, with lg1-mutant having two exons. The lg1-mutant harboured an insertion of 130 bp Tourist MITE transposable element in exon-2 at 1663rd base, which deleted 157 amino acids of C-terminal region of the mutant LG1 protein. The mutant LG1 protein was 247 amino acids in length, in contrast to 399-404 amino acids in wild-type protein. The analysis with 26 paralogues and 66 orthologues of Lg1 revealed conservation of the squamosa promoter-binding (SBP) domain. A PCR-based co-dominant InDel marker (MGU-lg1-Tourist) specific to insertion of 130 bp was developed that differentiated the mutant allele (lg1) from the wild-type allele (Lg1). The marker was validated in two F2 populations, which showed a 1:2:1 ratio. F2 plants showed a 3 (wide angle: 41.58°) :1 (narrow angle: 5.88°) segregation for leaf angle. A set of 11 gene-based InDel markers (MGU-InDel1 to MGU-InDel11) specific to Lg1 was also developed, and along with MGU-lg1-Tourist, they classified a diverse set of 48 inbreds into 35 distinct haplotypes (hap1 to hap35) with lg1-based inbreds possessing hap1. This is the first report of the development and validation of a co-dominant gene-based marker specific to lg1, and information generated assumes great significance in maize breeding aimed to tailor the plant architecture suitable for high plant density..

Zea mays↗
PubMed2025

Engineered virus-like particle-assembled Vegfa-targeting Cas9 ribonucleoprotein treatment alleviates neovascularization in wet age-related macular degeneration.

BACKGROUND: Age-related macular degeneration, particularly the wet form, is a leading cause of vision loss, characterized by vascular endothelial growth factor A (VEGFA) overproduction. Engineered virus-like particles (eVLPs) combine the efficiency of viral systems with the transient nature of non-viral platforms to offer a potential solution for delivering VEGFA-targeting genome editing enzymes in a safe and efficient manner. Here, we investigate the therapeutic efficacy of eVLPs for transient delivery of Vegfa-targeting Cas9 ribonucleoprotein in a laser-induced choroidal neovascularization mouse model of wet age-related macular degeneration. RESULTS: We find that Cas9-eVLPs enables efficient intracellular delivery in vitro, achieving up to 99% insertion and deletion frequency at Vegfa target locus and significant VEGFA protein downregulation in NIH/3T3 cells. A single subretinal injection of Cas9-eVLPs into the mouse retinal pigment epithelium effectively disrupts Vegfa expression, achieving an average indel efficiency of 16.7%. Compared to control groups, the laser-induced choroidal neovascularization mouse model exhibits significantly reduced choroidal neovascularization formation following Cas9-eVLPs intervention, and decreased VEGFA protein levels are detected in the retinal pigment epithelium. Furthermore, the retinal anatomical and functional toxicity are not affected after treatment. CONCLUSIONS: eVLPs exhibit the potential as a safe and efficient delivery platform for Cas9 ribonucleoproteins, achieving precise Vegfa downregulation and significant reduction in choroidal neovascularization in a mouse model of wet age-related macular degeneration. With transient delivery of gene editing enzymes, high editing efficiency, and minimal risk of genomic integration, eVLPs present a promising alternative to conventional delivery systems for advancing genome editing therapies in retinal diseases.

CRISPR-Associated Protein 9↗
PubMed2025

Indel mutation in transcription factor PabHLH2 regulates amygdalin accumulation and kernel bitterness in apricot.

Amygdalin, the phytochemical responsible for the characteristic bitterness of apricot (Prunus armeniaca L.) kernels, also exhibits significant bioactive properties and therapeutic potential. Genetic regulation of amygdalin content is therefore a key objective in apricot breeding programs aimed at quality improvement. In this study, we conducted quantitative trait loci (QTL) mapping to uncover the genetic basis of sweet-bitter differentiation in apricot kernels. We identified a 15-bp insertion/deletion (indel) polymorphism strongly related to kernel bitterness, with marker validation achieving 100% concordance across 601 apricot germplasm accessions. Notably, this polymorphic site is located within the helix-loop-helix (HLH) domain of the basic HLH (bHLH) transcription factor PabHLH2. Protein interaction analyses revealed that the 15-bp deletion variant impaired dimerization capacity, reducing transcriptional activation of downstream targets. Using yeast one-hybrid screening and dual-luciferase reporter assays, we identified PaCYP71AN24 and PaCYP79D16 as direct transcriptional targets of PabHLH2. Functional characterization further indicated that the PabHLH2a variant (harboring the 15-bp insertion) significantly enhanced the promoter activity of these cytochrome P450 genes compared with the deletion variant. Transient overexpression and silencing experiments in apricot kernels further confirmed that the 15-bp insertion positively regulates both PaCYP71AN24/PaCYP79D16 expression and prunasin accumulation, the immediate biosynthetic precursor of amygdalin. Overall, these findings provide mechanistic insights into the allelic variation underlying kernel bitterness and delineate the molecular cascade of amygdalin biosynthesis. The identified molecular markers and functional characterization establish a basis for marker-assisted breeding of low-amygdalin apricot cultivars, supporting the dual-purpose utilization of kernels in food and pharmaceutical industries.

Amygdalin↗
PubMed2026

Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.

Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.

CRISPR-Cas9↗
PubMed2026

MRE11 suppresses germline mutagenesis at meiotic double-strand breaks in mice.

SPO11 forms hundreds of double-strand breaks (DSBs) to initiate meiotic recombination that is normally error-free. However, SPO11 activity can be mutagenic when one chromatid incurs closely spaced DSBs (double cuts), especially when DSBs are dysregulated by loss of the ATM kinase. De novo indels and structural variants can arise via end joining at double cuts within a single hotspot (microdeletions) or at adjacent hotspots separated by at least 30 kb, as we now show, sometimes accompanied by ectopic insertions of double-cut fragments. Here, we investigate how meiotic DSB end processing influences end joining. In MRE11-deficient mouse spermatocytes, which do not resect their DSBs, deletions at double cuts occur readily, with end-joining breakpoint profiles closely matching SPO11 DSB profiles. Microdeletions suggest that two DSBs can be as close as ∼21 bp. The tyrosyl-DNA phosphodiesterase TDP2 contributes to both deletion formation and ectopic insertion of double-cut fragments, presumably by removing SPO11 from DNA ends prior to joining. Finally, observations suggest a cooperative role for MRE11 and ATM in locally regulating DSB distributions. Our findings provide insight into the mechanism of de novo mutation origin, emphasizing the role of meiotic DSBs in shaping genome evolution.

Animals↗
PubMed2025

The mutation landscape of Daphnia obtusa reveals evolutionary forces shaping genome stability.

Spontaneous mutations are the primary source of genetic variation and play a central role in shaping evolutionary processes. To investigate mutational dynamics in Daphnia obtusa, we generated a chromosome-level genome assembly spanning 129.4 Mb across 12 chromosomes, encompassing 15,321 predicted protein-coding genes. Leveraging whole-genome sequencing of eight mutation accumulation (MA) lines propagated for an average of 482 generations (spanning over 20 years), we estimated a spontaneous single nucleotide mutation (SNM) rate of 2.23 × 10-9 and an indel mutation rate of 2.75 × 10-10 per site per generation. The SNM spectrum was strongly biased toward C:G > T:A transitions. Comparative analyses with natural population data revealed that exonic mutations observed in the MA lines were significantly less likely to be present in standing variation than intronic or intergenic mutations, suggesting that purifying selection in natural populations acts to remove deleterious alleles. We also identified 48 de novo loss-of-heterozygosity (LOH) events, comprising 8 heterozygous deletions and 40 gene conversion events. The genome-wide gene conversion rate was estimated at 2.62 × 10-5 per heterozygous site per generation. These findings provide a comprehensive view of the mutation spectrum, selective pressures, and mechanisms underlying genome stability in D. obtusa.

Daphnia obtusa↗
PubMed2026

Features affecting Cas9-induced editing efficiency and patterns in tomato: evidence from a large CRISPR dataset.

CRISPR/Cas9 is a cornerstone of plant genome editing, yet the determinants of editing efficiency for a given single-guide RNAs (sgRNAs) and DNA double-strand break (DSB) repair outcomes remain poorly understood, particularly in plants. Here, we generated a large experimental dataset comprising 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts, and quantified editing efficiency and repair footprints together with chromatin accessibility and transcriptional state in the same cellular context. Editing efficiency was consistently higher at targets in accessible chromatin and modestly higher in promoters and introns than in exons, whereas transcriptional activity had no detectable effect. Editing efficiencies were more similar among sgRNAs targeting the same gene than among different genes, revealing a local genomic influence on Cas9 activity. A distinct subset of sgRNAs achieved near-complete editing and produced characteristic repair footprints dominated by long deletions with extended microhomology tracts, indicative of microhomology-mediated end joining (MMEJ), resembling patterns associated with high-efficiency guides in human cells, and suggesting conserved sequence-driven repair biases across species. In contrast, widely used human-trained prediction models failed to accurately rank sgRNA performance in plants, highlighting the limits of cross-species predictability. Together, this dataset provides a resource for improving guide design and mechanistic understanding of plant DNA repair.

Solanum lycopersicum↗
PubMed2026

Pangenomes aid accurate detection of large insertions and deletions from targeted sequencing: the case of cardiomyopathies.

BACKGROUND: Gene panels represent a widely used strategy for genetic testing in a vast range of Mendelian disorders. While this approach aids reliable bioinformatic detection of short coding variants, it often fails to detect many larger variants. Recent studies have recommended the adoption of pangenome references (as opposed to linear reference genomes like GRCh38) to augment detection of large variants from targeted sequencing, potentially providing diagnostic laboratories with the possibility to streamline diagnostic work-ups and reduce costs. METHODS: Here, we analyze 1969 cardiomyopathy cases and 1805 controls sequenced with the Illumina Trusight Cardio panel using a pangenome-based workflow (GRAF) and five conventional orthogonal methodologies (GATK HaplotypeCaller, GATK-gCNV, ExomeDepth, Manta and Lumpy-SV) to detect variants ≥ 20 bp in size. RESULTS: Following lab-based variant validation by means of PCR and Sanger sequencing, we show that GRAF conjugates higher precision and recall (F1 score 0.86) compared with other methods (F1 0-0.57) in detecting potentially pathogenic variants ≥ 20 bp from short-read panel data. Results were complemented by a comparison of the tools' performance in detecting ground truth variants on reference sample HG002 from Genome In A Bottle, which confirmed GRAF to outperform other tools also on exome sequencing (F1 0.97 vs. 0-0.94). Notably, in the HG002 benchmark dataset, GRAF also showed slightly improved performance compared to GATK HaplotypeCaller in the identification of small variants (1-19 bp; F1 0.975 vs. 0.968). CONCLUSIONS: Our results indicate that pangenome-based workflows aid improved detection of large variants from targeted sequencing data in the clinical context and suggest that they may contribute to more unified variant detection frameworks for all-size genetic variants in the future.

Humans↗