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PubMed2005

Spectral editing based on selective excitation and Lee-Goldburg cross-polarization under magic angle spinning.

We show that a Gaussian-shaped pulse can be used to excite selected 1H signals in hydroxyapatite, monetite and H-Y zeolite loaded with trimethylphosphine oxide (TMPO). This selective excitation method can be incorporated into Lee-Goldburg (LG) cross-polarization to obtain useful spectral editing opportunity. This new strategy has been applied to identify the Brønsted and the Lewis acid sites in H-Y zeolite using TMPO as the probe molecule.

Algorithms↗
PubMed2025

A streamlined base editor engineering strategy to reduce bystander editing.

Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, we show that integrating a naturally occurring oligonucleotide binding module into the deaminase active center of TadA-8e, a highly active deoxyadenosine deaminase, enhances its editing specificity. When conjugated with a Cas9 nickase or alternative PAM Cas9 variants, the engineered TadA variant-TadA-NW1-consistently achieves robust A-to-G editing efficiencies within an editing window consisting of four nucleotides, substantially narrower than the 10-bp editing window of the TadA-8e-derived ABEs. Moreover, compared to ABE8e, ABE-NW1 shows significantly decreased Cas9-dependent and -independent off-target activity while maintaining similar on-target editing efficiency. Further, TadA-NW1 can be reprogrammed to perform desired cytidine deamination and adenine transversion within a restricted editing window. Finally, in a cystic fibrosis (CF) cell model, ABE-NW1 outperforms existing ABEs in accurately and efficiently correcting the CFTR W1282X variant, one of the most common CF-causing mutations. In all, we engineered a suite of base editors with refined activity windows, enabling more precise base editing. Importantly, this study presents a streamlined genome editor re-engineering strategy to accelerate the development of therapeutic base editing.

Gene Editing↗
PubMed2005

Resolution-enhanced base-type-edited HCN experiment for RNA.

New base-type-edited transverse-relaxation optimized CT-HCN(C) experiments are presented that yield intra-base and sugar-to-base correlations for 13C-15N labeled RNA. High spectral resolution in the 13C and 15N dimensions is achieved by constant time (CT) frequency editing. A spectral editing filter applied during the CT 15N labeling period separates the correlation peaks arising from G/U and A/C nucleotide bases. This provides the increased spectral resolution required to unambiguously connect the 1H and 13C resonances in sugar and base moieties of RNA nucleotides. In addition, the experiment allows base type identification for each residue, and therefore presents an attractive spectroscopic alternative to nucleotide-specific isotope labeling. Application to a 33-nucleotide RNA aptamer demonstrates the performance of the new pulse scheme.

Carbon Isotopes↗
PubMed2025

Engineering and comparison of cas12a-based genome editing systems in plants.

While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.

Gene Editing↗
PubMed2026

Miniature and versatile genome regulation TnpB-ωRNA toolkits facilitate cancer immunotherapy.

CRISPR‒Cas systems represent powerful tools for genome regulation. However, the large size of Cas proteins limits their efficient delivery via an adeno-associated virus (AAV), thereby restricting their clinical translation. Here, we engineer the IS200/IS605 transposon-encoded nuclease TnpB, along with its ωRNA scaffold, to create an enhanced TnpB system, which serves as a compact toolkit for gene activation, genome editing, and base editing. The gene activator enTnpBa increases expression by 2889-fold with a minimized 93 nt ωRNA and robustly activates endogenous genes in mammalian cells. We develop a single-AAV-based regimen for immune activation (AAV-ImmunAct) that delivers enTnpBa to activate CXCL9, IL-15, and IFN-γ. AAV-ImmunAct effectively enhances T cell migration and activation, increases killing of cancer cell lines and patient-derived organoids, and synergizes with anti-PD-1 therapy in humanized mice. Here, we establish enTnpB as a compact and versatile platform for genome regulation and a promising tool for cancer immunotherapy.

Humans↗
PubMed2026

Transforming Plant Viruses into Vectors for Next-Generation Agriculture-A Review.

Plant viral vectors have evolved from tools for transient gene expression into a versatile platform for precise genetic intervention, offering a rapid, transgene-free alternative to conventional crop transformation. This review critically assesses their engineering for scalable field application, moving beyond foundational techniques like virus-induced gene silencing (VIGS). We highlight how advanced vector design, including deconstructed genomes and synthetic regulatory circuits enhances cargo capacity, specificity, and biosafety. The integration of viral delivery with CRISPR-Cas systems has unlocked virus-induced genome editing (VIGE), base editing, and prime editing, enabling heritable trait modification without tissue culture. However, the transition from proof-of-concept in model plants to robust field technology hinges on overcoming critical bottlenecks: expanding host range through chimeric vectors, ensuring environmental containment, and developing scalable delivery methods such as nano-formulations or adjusted agroinfiltration protocols. We evaluate these delivery routes and emerging synergies with nanobiotechnology for targeted and efficient applications. While challenges in regulation, public perception, and large-scale production persist, the strategic engineering of viral vectors for stability, specificity, and safety positions them as a transformative, next-generation biotechnological input for achieving sustainable crop improvement and protection under changing climatic conditions.

Plant Viruses↗
PubMed2000

An AU-rich sequence element (UUUN[A/U]U) downstream of the edited C in apolipoprotein B mRNA is a high-affinity binding site for Apobec-1: binding of Apobec-1 to this motif in the 3' untranslated region of c-myc increases mRNA stability.

Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a K(d) of approximately 435 nM. A series of AU-rich templates was used to identify a high-affinity ( approximately 50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3' untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA-specific cytidine deaminase.

3' Untranslated Regions↗
PubMed2026

Engineered Transformer Base Editor with Enhanced Editing Efficiency.

Canonical cytosine base editors (CBEs) achieve precise C-to-T conversions without inducing DNA double-strand breaks (DSBs), yet their clinical potential remains hampered by substantial off-target (OT) mutations. The recently developed transformer base editor (tBE) significantly reduces both genomic and transcriptomic OT mutations by using a cleavable deoxycytidine deaminase inhibitor (dCDI) domain. However, the modest base editing efficiency limits its broader applications. Here, through rational deaminase engineering and fusion of a uracil DNA glycosylase inhibitor (UGI) domain, we developed the engineered tBE (etBE). The etBE exhibited substantially enhanced editing efficiencies compared with the parental tBE (up to 35.11-fold improvement), while maintaining high editing fidelity and background levels of OT mutations. As a therapeutic proof-of-concept, dual adeno-associated virus (AAV)-mediated delivery of etBE targeting proprotein convertase subtilisin/kexin type 9 (PCSK9), a well-established therapeutic target for cardiovascular diseases, was evaluated in a humanized mouse model. The treatment achieved efficient in vivo base editing (up to 35.13%), resulting in substantial reductions in plasma PCSK9 protein (24%) and low-density lipoprotein cholesterol (LDL-C) levels (33%), while inducing only minimal OT mutations. Collectively, etBE represents a highly efficient and specific base editing platform with enormous potential for both basic research and clinical applications.

CRISPR‐Cas9↗
PubMed2026

The present and future of nonviral delivery-based genome editing for hereditary hearing loss.

PURPOSE OF REVIEW: This review summarizes nonviral genome-editing delivery platforms for hereditary hearing loss, focusing on lipid nanoparticles (LNPs) and engineered virus-like particles (eVLPs), and discusses their advantages over adeno-associated virus-based delivery, as well as the barriers to clinical translation. RECENT FINDINGS: Recent advances have established LNPs as a clinically advanced nonviral platform, although challenges related to inner ear biodistribution, cell type specificity, endosomal escape, and immunogenicity remain to be addressed. In parallel, eVLPs have undergone substantial technical evolution, progressing from early low efficiency systems to advanced base editor- and prime editor-eVLP architectures that enhance cargo loading and editing efficiency. Extracellular vesicle-based genome editing has also emerged as an additional platform, although issues related to reproducibility, loading efficiency, and scalability remain major hurdles. SUMMARY: Nonviral genome editing platforms expand the therapeutic toolkit for hereditary hearing loss by enabling transient delivery of genome editors with potential safety advantages. Future efforts should focus on characterizing biodistribution and immunogenicity, refining cell type-specific tropism, and establishing scalable manufacturing processes to enable successful clinical translation.

Humans↗
PubMed2025

An advanced cytosine base editor enabled the generation of cattle with a stop codon in the β-lactoglobulin gene.

β-Lactoglobulin (BLG) is an allergen present in milk that can induce an acute immune response in certain individuals. The successful use of cytosine base editors (CBEs) can introduce stop codons into premature mRNA, thereby generating animals with disrupted genes that negatively regulate target traits. In this study, we employed a CBE system to target the major milk allergen BLG in bovine embryos, mammary epithelial cells, and live cattle. First, the precise single-base editing of the BLG gene in bovine embryos was achieved by designing an effective sgRNA to induce a c.61C > T substitution in the coding region, converting codon 21Gln (p.21Gln) to a premature stop codon. Sanger sequencing revealed an editing efficiency of 83.3% (20 out of 24 embryos), including two homozygous edits. Second, a bovine mammary epithelial cell line harboring BLG edits was constructed using the same CBE system. Sequencing showed that the designed sgRNA1 enabled the simultaneous conversion of three consecutive cytosines (c.59-61CCC > TTT) to thymines. At position c.61, single-cell clones exhibited monoallelic or biallelic editing (BLGc.61C > T), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLGc.61C > T mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLGc.61C > T single-base-edited dairy cow that despite its heterozygosity, showed significantly reduced BLG expression in the mammary epithelial cells and milk. Collectively, this study demonstrates the feasibility of using CBEs to disrupt BLG expression in dairy cows and provides a foundation for application in generating hypoallergenic dairy products.

Animals↗
PubMed2025

Stacked mutations in multi-copy AHAS genes enhance sulfonylurea herbicide resistance in soybean.

Weeds are a major factor that negatively impact crop yields. Developing herbicide-resistant germlines is crucial for efficient weed control. Sulfonylurea- and pyrimidinyl benzoate-based herbicides inhibit the function of acetohydroxyacid synthase (AHAS), a key enzyme in the biosynthesis of branched-chain amino acids in plants. To create soybean plants resistant to these classes of herbicides, we performed base editing of AHAS genes in Glycine max. A guide RNA was designed to target the codon for proline-182 in GmAHAS2, with the prediction that off-target base editing might also occur in the GmAHAS3 and GmAHAS4 genes. We selected six genome-edited soybean lines, each carrying distinct mutations in GmAHAS2, GmAHAS3, or GmAHAS4. These lines were treated with three different AHAS-targeting herbicides to evaluate resistance. The results show that the number of mutated GmAHAS genes and the mutation patterns significantly influence herbicide resistance.

Herbicide Resistance↗
PubMed1996

RNA editing of glutamate receptor subunits GluR2, GluR5 and GluR6 in transient cerebral ischemia in the rat.

The mechanisms of ischemic cell damage are still not fully understood. It has been shown that alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)/kainate receptor antagonists, such as 6-nitro-7- sulphamoyl-benzo-(f)-quinoxaline-2, 3-dione (NBQX), are neuroprotective in models of transient forebrain ischemia, even when applied during recovery, indicating that nonNMDA receptors may play a pivotal role in ischemic cell damage. In the present series of experiments, we studied whether transient cerebral ischemia causes changes in the extent of mRNA editing of AMPA/kainate receptor subunits, a reaction critical for the control of calcium flux through nonNMDA receptor ion channels. Transient cerebral ischemia was produced in rats using the four-vessel occlusion (4-VO) model. After 30 min of ischemia, brains were recirculated for 4, 8, or 24 h. Total RNA was extracted from the cortex, striatum, and hippocampus in order to analyze the extent of mRNA editing of the glutamate receptor subunits GluR2, GluR5, and GluR6. RNA was converted by reverse transcription into cDNA, which was used as a template for subunit-specific polymerase chain reaction (PCR) to amplify a product across the edited base A (A edited to I in the second transmembrane-spanning regions of GluR2, GluR5, and GluR6). PCR products were analyzed with the restriction enzyme Bbv 1, which recognizes the cDNA sequence GCAGC originating from unedited but not that originating from edited GluR2, GluR5, or GluR6 mRNA (GCGGC, the base I is read as G). Restriction digests were electrophoresed, and the bands visualized with ethidium bromide and then photographed. The extent of mRNA editing of the different subunits was quantified using image analysis and appropriate standards. In all control brains studied, GluR2 mRNA was completely edited and remained so after reversible cerebral ischemia. The extent of GluR5 mRNA editing was significantly upregulated in the striatum (from 39 +/- 6% in controls to 57 +/- 9 and 56 +/- 7 after 4 and 8 h of recovery, respectively, p < 0.05 versus control) but not in the cortex and hippocampus. The extent of GluR6 mRNA editing was significantly reduced after 24 h of recovery: in the cortex, from 92 +/- 1 to 78 +/- 6% (p < 0.01); in the striatum, from 91 +/- 2 to 79 +/- 1% (p < 0.001); and in the hippocampus, from 90 +/- 3 to 80 +/- 2% (p < 0.05). A significant reduction was already apparent in the striatum after 4 h of recovery (p < 0.05). Results indicate that mRNA editing is regulated differently in each of the glutamate receptor subunits GluR2, GluR5, and GluR6 after transient cerebral ischemia. The ischemia-induced upregulation of GluR5 mRNA editing observed in the striatum may be indicative of a higher sensitivity to transient ischemia of neurons that exhibit a large fraction of unedited GluR5 mRNA. This assumption is corroborated by the observation (Mackler and Eberwine, 1993) that GluR5 mRNA is completely unedited in neurons of the hippocampal CA1-subfield, a region most vulnerable to transient cerebral ischemia. Whether the decrease in GluR6 mRNA editing observed in all brain structures after ischemia results from a disturbance of the editing reaction or from glial proliferation will have to be established in further experiments. Studying ischemia-induced changes in mRNA editing of glutamate receptor subunits GluR5 and GluR6 may help to elucidate the molecular mechanisms of ischemic cell damage.

Animals↗
PubMed1992

Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B cDNA constructs.

Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins.

Amino Acid Sequence↗
PubMedDate not supplied

Time lines and computer-based visual editing: new techniques for assessing exposure in epidemiologic studies.

We describe a time-line-based methodology for collecting exposure data for epidemiologic studies and for processing these data for statistical analysis with readily available software for the personal computer. The four components to this approach are: (1) collecting data in a memory-enhancing time-line format; (2) entering data from time lines into a computer database and editing them; (3) making a quantitative estimate of exposure, intake, or dose for each exposure event; and (4) creating analysis datasets by 'slicing' the quantified time lines based on desired exposure intervals or disease latent periods. Compared with fixed-format interviews, time-line-based interviews help subjects organize remembered events, thereby reducing confusion. They do not restrict responses to predetermined categorical exposure responses. The time-line methodology also facilitates the collection of supplementary data necessary for computing doses for complex exposures and the packaging of quantified exposures into analysis datasets for any time period of interest.

Child↗
PubMed2001

C-->U editing of neurofibromatosis 1 mRNA occurs in tumors that express both the type II transcript and apobec-1, the catalytic subunit of the apolipoprotein B mRNA-editing enzyme.

C-->U RNA editing of neurofibromatosis 1 (NF1) mRNA changes an arginine (CGA) to a UGA translational stop codon, predicted to result in translational termination of the edited mRNA. Previous studies demonstrated varying degrees of C-->U RNA editing in peripheral nerve-sheath tumor samples (PNSTs) from patients with NF1, but the basis for this heterogeneity was unexplained. In addition, the role, if any, of apobec-1, the catalytic deaminase that mediates C-->U editing of mammalian apolipoprotein B (apoB) RNA, was unresolved. We have examined these questions in PNSTs from patients with NF1 and demonstrate that a subset (8/34) manifest C-->U editing of RNA. Two distinguishing characteristics were found in the PNSTs that demonstrated editing of NF1 RNA. First, these tumors express apobec-1 mRNA, the first demonstration, in humans, of its expression beyond the luminal gastrointestinal tract. Second, PNSTs with C-->U editing of RNA manifest increased proportions of an alternatively spliced exon, 23A, downstream of the edited base. C-->U editing of RNA in these PNSTs was observed preferentially in transcripts containing exon 23A. These findings were complemented by in vitro studies using synthetic RNA templates incubated in the presence of recombinant apobec-1, which again confirmed preferential editing of transcripts containing exon 23A. Finally, adenovirus-mediated transfection of HepG2 cells revealed induction of editing of apoB RNA, along with preferential editing of NF1 transcripts containing exon 23A. Taken together, the data support the hypothesis that C-->U RNA editing of the NF1 transcript occurs both in a subset of PNSTs and in an alternatively spliced form containing a downstream exon, presumably an optimal configuration for enzymatic deamination by apobec-1.

APOBEC-1 Deaminase↗