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PubMed2026

Turnip Mosaic Virus-Based gRNA Delivery System for Plant Genome Editing.

Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE (NbPDS) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial endoribonuclease Csy4 increased editing efficiencies in the two NbPDS genes, raising editing in infiltrated leaves to 7.1%-13.8% for NbPDSa and 7.6%-23.0% for NbPDSb, whereas lower but reproducible editing was detectable in systemic leaves. The TuMV-Csy4 platform also supported editing of a second endogenous target, MAGNESIUM CHELATASE SUBUNIT H (NbChlH), and enabled multiplex editing of NbPDS and NbChlH regardless of guide order. Editing efficiencies were consistently higher in infiltrated leaves than in systemic leaves, and no visible photobleaching or chlorosis was observed in systemic tissues despite confirmed molecular editing. To assess the potential for heritable editing, a tRNAIle mobility element was fused to the NbPDS gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, demonstrating that heritable editing was not achieved under these conditions or did not result in mutations in all copies of the two NbPDS genes. Together, these results establish TuMV as a platform for Cas9-based gRNA delivery and show that Csy4-mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus-based genome editing systems in plants.

genome editing platform↗
PubMed1993

RNA editing of a chimeric maize mitochondrial gene transcript is sequence specific.

RNA editing was analysed in the mitochondrial ATPase complex subunit 6 gene (atp6) transcripts of the C male-sterile cytoplasm (cms-C) of maize. The only copy of atp6 in cms-C, designated C-atp6, is a triple gene fusion product comprised of DNA sequences derived from atp9, atp6, and an unknown origin. Sequences of cDNAs revealed 19 C to U alterations resulting in 16 amino acid residue changes compared to the genomic sequence. The only C to U edit in the 39-nucleotide sequence similar to atp9 was comparable to a change in the complete atp9 mRNAs of Petunia, Oenothera, wheat, and sorghum. The 442 nucleotides of unknown origin were not edited. The 18 editing events within the atp6 homologous region were similar to those in the atp6 transcripts of sorghum. RNA editing in maize C-atp6 transcripts introduces a translational stop codon at the same position where it is created by editing in sorghum and Oenothera atp6 mRNAs and is already present in atp6 open reading frames of most other plant and non-plant organisms. Our results, along with other reports on editing in chimeric transcripts, indicate that RNA editing is not influenced by rearrangements but instead is sequence specific.

Adenosine Triphosphatases↗
PubMed1991

The protein-encoding gene T-urf13 is not edited in maize mitochondria.

RNA editing of T-urf13, a gene specific to the mitochondria of cytoplasmic male-sterile, type-T (cms-T) maize, and an adjacent, cotranscribed gene orf221, have been studied by cDNA sequencing. No editing was detected in 22 cDNA clones. This is the only report of a polypeptide-encoding gene in higher-plant mitochondria that is not edited. T-urf13 may not be edited because it is derived largely from the coding and flanking regions, which are rarely edited, of a ribosomal RNA gene. orf221 is edited; however, the similarity between the predicted amino acid sequences of orf221 in cms-T and normal cytoplasms is not increased.

Amino Acid Sequence↗
PubMed2002

[Study on the editing sites of the coxII gene transcripts of rice mitochondria].

The term RNA editing is generally used to describe those molecular processes in which the information content is altered in an RNA molecule. This process is not limited to mRNA since alterations of non-informational RNA have also been found. RNA editing exists extensively in the higher plant mitochondria, and is the necessary step for forming functional proteins. In this paper, the research materials are the gametopthyte male sterility line (A), maintainer line (B) and F1 hybrid (F1) of HL-type CMS. 15 editing sites are found in the transcripts of coxII by comparing cDNAs and DNAs sequences. A,B and F1 have same Editing sites. When editing occurs at the first or second position of codons, the encoded amino acid is likely to be altered. As a result, the conservation of the predicted protein is improved as compared with other organisms.

Base Sequence↗
PubMed2025

RNA-DNA hybrid binding domain broadens the editing window of base editors.

Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.

Gene Editing↗
PubMed1995

Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids.

In tobacco plastids, functional psbL mRNA is created by editing an ACG codon to an AUG translation initiation codon. To determine if editing may occur in a chimeric mRNA, the N-terminal part of psbL containing the editing site was translationally fused with the aadA and kan bacterial genes. The chimeric constructs were introduced into the tobacco plastid genome by targeted gene insertion. Editing of the chimeric mRNAs indicated that the 98 nt fragment spanning the psbL editing site contains all cis information required for editing. Expression of the chimeric gene transcripts led to a significant decrease in the editing efficiency of the endogenous psbL mRNA. However, the efficiency of editing in the transplastomic lines was unchanged for four sites in the rpoB and ndhB mRNAs. Reduced efficiency of psbL editing, but not of the other four sites, in the transplastomic lines indicates depletion of psbL-specific editing factor(s). This finding implicates the involvement of site-specific factors in editing of plastid mRNAs in higher plants.

Amino Acid Sequence↗
PubMed2026

Development and application of a fast and efficient CRISPR/Cas12f -based genetic toolkit in Bacillus cereus GW-01.

Bacillus cereus GW-01, an efficient degrader of β-cypermethrin (β-CY), has a high safety profile and probiotic potential for regulating intestinal flora and fermented foods, which is difficult to genetically engineer for modification due to its restrictive modification system. This study successfully developed a CRISPR/enCas12f-based genome editing system, first selecting the plcR gene for proof-of-concept validation with 100% knockout efficiency. Subsequently, this system was utilized to delete the virulence gene nheABC in GW-01, yielding a safer probiotic strain. Compared with the wild-type strain GW-01, the probiotic-related indicators of the ΔnheABC mutant, including cell surface hydrophobicity, auto-aggregation ability and biofilm formation ability, were 80%, 90% and 2.9 (OD₅₉₅), respectively. There were no significant differences in these indicators between the mutant and the wild type. Meanwhile, the ΔnheABC mutant still maintained a high β-cypermethrin degradation efficiency of 80% at the concentration of 30 μg/mL. This work facilitates functional genomic research and genetic modification of Bacillus cereus GW-01. The established CRISPR/enCas12f system enables targeted gene deletion to explore gene functions and phenotypic mechanisms, and paves the way for its development into safe probiotics and excellent microbial chassis.

Bacillus cereus↗
PubMed2026

CRISPR-Cas technologies for precision genome editing in plants: advances, applications, and future perspectives.

Developing climate-smart crops with enhanced crop productivity, nutritional quality, resistance to biological and environmental stressors is vital for global food security. While hybrid breeding forms the cornerstone of modern crop improvement, conventional breeding approaches are limited by genetic barriers and prolonged breeding cycles. CRISPR-Cas based genome editing has revolutionized plant biology by allowing precise, efficient, and multiplex genetic modifications. This review provides a comprehensive synthesis of a recent advances in CRISPR-Cas technologies and their strategic applications in crop genetics and hybrid breeding. We summarize major genome-editing strategies, including gene knock-out, base editing (BE), knock-in, gene replacement, epigenome editing, and transcriptional regulation. Furthermore, we contrast stable, transient, and DNA-free delivery systems, highlighting ribonucleoprotein (RNP)-mediated delivery for minimizing off-target effects and avoiding transgene integration. We showcase how these technologies accelerate hybrid breeding by engineering male sterility systems, fixing heterosis, and generating high-throughput mutant libraries for trait discovery. Finally, we synthesize major bottlenecks in tissue culture-independent transformation and delivery systems, while outlining how emerging paradigms like de novo domestication and synthetic biology will shape the future of climate-resilient agriculture.

CRISPR/Cas↗
PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗
PubMed1994

Dimeric structure of a human apolipoprotein B mRNA editing protein and cloning and chromosomal localization of its gene.

Apolipoprotein B (apoB) mRNA editing consists of a posttranscriptional C-->U conversion involving the first base of the codon CAA encoding glutamine-2153 to UAA, a stop codon, in apoB mRNA. Using a cloned rat cDNA as a probe, we cloned the cDNA and genomic sequences of the gene for a human apoB mRNA editing protein. Expression of the cDNA in HepG2 cells results in editing of the intracellular apoB mRNA. By fluorescence in situ hybridization, we localized the gene for the editing protein to chromosome band 12p13.1-p13.2. By Northern blot analysis, it was shown that the human editing protein mRNA is expressed exclusively in the small intestine. The cDNA sequence predicts a translation product of 236-aa residues. By attaching an epitope tag sequence to the C terminus of the editing protein, we examined the polymerization state of the editing protein synthesized in vitro. We found that the editing protein undergoes spontaneous polymerization. The migration of the human apoB mRNA editing protein on an HPLC column and the stoichiometry of polymeric epitope-tagged to untagged protein indicate that the protein exists as a dimer. Dimerization does not require glycosylation of a consensus N-linked glycosylation sequence present in the protein and is not mediated by disulfide bridge formation. The human apoB mRNA editing protein is a cytidine deaminase showing structural homology to some known mammalian and bacteriophage deoxycytidylate deaminases. The latter enzymes exist as homopolymers. The fact that the apoB mRNA editing protein also exists as a homodimer has important implications for the mechanism of apoB mRNA editing in humans.

APOBEC-1 Deaminase↗
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↗
PubMed2026

The application of the CRISPR-Cas system in Pseudomonas aeruginosa infections.

Due to the extensive drug resistance of Pseudomonas aeruginosa (P. aeruginosa), it is still a great clinical challenge. The clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) system has become a promising strategy against this pathogen. This review critically evaluates the multifaceted applications of CRISPR-Cas technology in P. aeruginosa, including its role in antimicrobial resistance, diagnostics, genome editing, and emerging therapeutic and vaccine strategies. In addition to conducting a comprehensive analysis of various studies, we also compared the performance and limitations of various CRISPR platforms, and discussed the main technologies and transformation obstacles in this field. Finally, we look forward to the direction of applying these experimental tools to clinical research in the future.

Pseudomonas aeruginosa↗
PubMed1994

RNA editing of transcripts of a chimeric mitochondrial gene associated with cytoplasmic male-sterility in Brassica.

The orf224 gene is a chimeric open reading frame associated with the Polima or pol cytoplasmic male sterility of Brassica napus. The first 58 codons and 5' upstream region of orf224 are derived from a conventional mitochondrial gene, orfB, while the origin of the remaining portion of the gene is unknown. Transcripts of the orf224 gene were found to be edited at a single site in the region of the gene that does not correspond to a known sequence. Oligonucleotides corresponding to the edited and unedited forms were shown to hybridize specifically to respective in vitro orf224 transcripts. Analysis of floral mtRNA by this method indicated that virtually all orf224 transcripts of both sterile and fertile, nuclear-restored pol cytoplasm plants are edited. Our results indicate that transcripts of novel, CMS-associated genes may be edited, but that, at least in this case, the degree of editing does not appear to be directly related to the male-sterile phenotype.

Amino Acid Sequence↗
PubMed1999

The ATPase subunit 6 gene sequence predicts that RNA editing is conserved between lizard- and human-infecting Leishmania.

Here we investigate the similarities in the kinetoplastid RNA editing process between human- and lizard-infecting Leishmania species. We present the sequence of the maxicircle-encoded ATPase subunit 6 gene from L. (V.) panamensis, L. (L.) mexicana and L. (L.) donovani species of human-infecting Leishmania. These represent the first available sequences of this gene from Leishmania species other than the lizard-infecting L. tarentolae. The gene sequences are highly conserved, both over the edited and unedited parts of the gene, implying that the RNA editing process is likely to be highly conserved between Leishmania species. Indeed, the first editing domain is absolutely conserved in all three Leishmania species studied and L. tarentolae. A phylogeny based on part of the ATPase subunit 6 gene placed the lizard-infecting Leishmania within the monophyletic Leishmania genus, supporting previous data which suggest that lizard- and human-infecting Leishmania species are closely related.

Adenosine Triphosphatases↗
PubMed1991

The Sendai virus P gene expresses both an essential protein and an inhibitor of RNA synthesis by shuffling modules via mRNA editing.

The P gene of Sendai virus expresses as many as eight proteins, two of which (V and W) are expressed only from edited mRNAs; only the P protein is known to be involved in RNA synthesis. To examine the functions of the other P gene proteins, we developed an in vivo system in which genome replication is driven by plasmid generated viral proteins. We found that P was essential for this process, whereas V and W were not only non-essential, they were inhibitory. By using various P gene deletions and varying the amounts of plasmids transfected, we provide evidence that P is a modular protein. The N-terminal domain (shared with V and W) binds the L or polymerase protein, whereas the C-terminal domain binds the nucleoprotein NP. A model of paramyxovirus RNA synthesis is presented, and the implications of negative regulation during persistent infection are discussed.

Blotting, Northern↗
PubMed2002

Transcription of succinate dehydrogenase subunit 4 (sdh4) gene in potato: detection of extensive RNA editing and co-transcription with cytochrome oxidase subunit III (cox3) gene.

The potato (Solanum tuberosum L.) mitochondrial cox3/ sdh4 gene cluster was previously identified by heterologous hybridization using a Marchantia polymorpha sdh4 probe. In this work, this potato gene cluster was cloned, sequenced and its expression was evaluated. The gene sequence and gene locus organization were found to be similar to the corresponding gene cluster in other dicot species, where known. Northern hybridizations with potato and cauliflower (Brassica oleraceae) mtRNA and RT-PCR analyses using potato mtRNA indicated that cox3 and sdh4 are co-transcribed in both species, generating a complex transcription pattern, where several transcripts from 1.1 kb to 4.4 kb are found. The potato transcript from this cluster displayed 14 and 13 RNA-editing sites, in the cox3 and sdh4 genes respectively, which changed the codon identity to amino acids and created a sdh4 partially edited stop codon. Forty-three cDNA clones were analyzed for editing process and revealed different partial-editing with no apparent sequential processing in the sdh4 gene.

Arabidopsis Proteins↗
PubMed1996

Protein polymorphism generated by differential RNA editing of a plant mitochondrial rps12 gene.

The rps12 gene transcripts encoding mitochondrial ribosomal protein S12 are partially edited in petunia mitochondria. Different petunia lines were found vary in the extent of rps12 transcript editing. To test whether multiple forms of RPS12 proteins are produced in petunia mitochondria as a result of partial editing, we probed mitochondrial proteins with specific antibodies against edited and unedited forms of a 13-amino-acid RPS12 peptide spanning two amino acids affected by RNA editing. Both antibodies reacted with mitochondrial proteins at the expected size for RPS12 proteins. The amounts of unedited RPS12 protein in different petunia lines correlate with the abundance of unedited transcripts in these plants. Unedited rps12 translation products are also detected in other plant species, indicating that polymorphism in mitochondrial rps12 expression is widespread. Moreover, we show that RPS12 proteins recognized by both edited-specific and unedited-specific antibodies are present in a petunia mitochondrial ribosome fraction. These results demonstrate that partially edited transcripts can be translated and that the protein product can accumulate to detectable levels. Therefore, genes exhibiting incompletely edited transcripts can encode more than one gene product in plant mitochondria.

Amino Acid Sequence↗