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At least 127 records · Page 7Linked to original sources

Diversity at the HYP1 locus in potato cyst nematodes does not result from developmentally-programmed somatic mutations.

Most genetic diversity stems from spontaneous mutations, that is, errors in DNA repair or replication. But for dozens of organisms across the tree of life, mutations at specific loci are not spontaneous but developmentally programmed: effectively, some organisms edit their own DNA sequences. This is perhaps most common among pathogens and parasites, many of which use editing to diversify genes that produce important antigens. Plant-parasitic potato cyst nematodes are damaging agricultural pests that establish a lifelong feeding site inside the root of their host plant. We previously observed extensive diversity of rare alleles at HYP1, the most highly expressed gene that encodes a protein secreted by potato cyst nematodes during parasitism. Importantly, HYP1 alleles differ from each other by complex, in-frame rearrangements of short repeated sequence motifs within a single exon. Combining several lines of evidence, we previously hypothesized that potato cyst nematodes use developmentally-programmed mutations, or editing, to diversify HYP1 alleles in the soma. In the current work, we now test this hypothesis. We employ highly accurate long-read DNA sequencing of a simplified genetic system to identify potential rare edited alleles, we use a transgenic yeast system to describe large de novo mutations at HYP1, and we interpret our findings in light of key population genetic parameters as well as the genetic diversity surrounding HYP1 and across the genome.

Animals↗

The chlB gene encoding a subunit of light-independent protochlorophyllide reductase is edited in chloroplasts of conifers.

ChlB is one of three chloroplast genes shown so far to be required for light-independent chlorophyll synthesis. It occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have demonstrated, for the first time in conifer chloroplasts, the presence of two internal C to U editing sites in this transcript. In the chlB transcript of Pinus sylvestris, the editing of the second position in a CCG codon leads to an amino-acid substitution from proline to leucine. Editing of a nearby CGG codon, resulting in an arginine to tryptophan substitution, has also been observed. The nucleotide sequence of this region has been compared with other species of gymnosperms. Out of seven species analysed, editing at both sites has only been detected in spruce, while in Larix only the editing which results in the Arg to Trp substitution was found. In other cases, both leucine and tryptophan are encoded by cpDNA, suggesting that conservation of these amino acids, through encoding by DNA or by editing of the RNA, is critical for the protein function. Transcripts are partially edited at the CGG codon and the relative abundance of cDNA molecules with the edited C is species-specific. The possible involvement of RNA editing in the regulation of gene expression in different organs of pine seedlings is discussed.

Amino Acid Sequence↗

Fate of a larch unedited tRNA precursor expressed in potato mitochondria.

In higher plant mitochondria, post-transcriptional C to U conversion known as editing mostly affects mRNAs. However, three tRNAs were also shown to be edited. Among them, three editing sites were identified in larch mitochondrial tRNA(His). We have previously shown that only the edited version can undergo maturation in vitro. In this paper, we introduced via direct DNA uptake the edited or unedited version of larch mitochondrial trnH into isolated potato mitochondria and expressed them under the control of potato mitochondrial 18 S rRNA promoter. As expected, the edited form of larch mitochondrial tRNA(His) precursor was processed in the isolated organelles. By contrast, no mature tRNA(His) was detected when using the unedited version of trnH. However, precursor molecules could be characterized by reverse transcription-PCR. These data demonstrate that the potato mitochondrial editing machinery is not able to recognize these "foreign" editing sites and confirm that these unedited tRNA precursor molecules are not correctly processed in organello. As a consequence, the fate of these RNA precursor molecules is likely to be degradation. Indeed, we detected by PCR two 3'-end truncated precursor RNAs. Interestingly, both RNA species exhibit poly(A) tails, a hallmark of degradation in plant mitochondria. Taken together, these data suggest that, in plant mitochondria, a defective unedited RNA precursor that cannot be processed to give a mature stable tRNA, is degraded through a polyadenylation-dependent pathway.

Animals↗

Development of a highly efficient protoplast regeneration and transfection protocol for enhancing CRISPR genome editing of Brassica carinata.

Brassica carinata is an important oil crop with significant potential for food and industrial production. The application of the CRISPR/Cas9 genome editing tool in B. carinata could accelerate its breeding cycle. However, no efficient DNA-free gene editing method currently exists for this species. Protoplast-based CRISPR editing presents a promising solution, though it is often challenging for many crop species. In this study, we investigated several critical factors influencing in vitro shoot regeneration, including genotype, sugar type, selection and combination of plant growth regulators (PGRs), and culture duration on different media throughout various stages of protoplast development. As a result, we developed a highly efficient, five-stage protoplast regeneration protocol for B. carinata based on specific stages of protoplast development. Key findings of this study include the requirement for high concentrations of NAA and 2,4-D in the initial medium (MI) for cell wall formation, while a lower auxin concentration relative to cytokinin was necessary for active cell division (MII). For callus growth and shoot induction, a high cytokinin-to-auxin ratio was essential (MIII), and an even higher cytokinin-to-auxin ratio was optimal for shoot regeneration (MIV). For shoot elongation, low levels of BAP and GA3 were sufficient (MV). Our results also demonstrated that the duration of culture on different media and maintaining appropriate osmotic pressure at the early stages were crucial for successful protoplast regeneration. With this optimized protocol, we achieved an average regeneration frequency of up to 64% and a transfection efficiency of 40% using the GFP marker gene. This efficient protoplast regeneration protocol is now being employed for genome editing in our lab and is expected to significantly enhance the application of the CRISPR system in both basic research and the genetic improvement of B. carinata over the long term.

Brassica carinata↗

Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing.

Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi syndrome (PWS) results from loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control the expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

Prader-Willi Syndrome↗

Enzymic editing mechanisms and the origin of biological information transfer.

Current knowledge of enzymic editing mechanisms in DNA replication, transcription and translation can be used to predict error rates in the absence of editing. Primitive enzymes which possessed synthetic activity but not yet editing mechanisms would have had extremely high error rates resulting in heterogeneous proteins. Based on present knowledge of molecular biology and biochemistry, it is concluded that the evolution of contemporary information transfer systems from primitive systems lacking such editing mechanisms remains an unsolved problem in theoretical biology.

Amino Acid Sequence↗

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↗

In Vivo Genome Editing Approach to Disrupt Hydroxyacid Oxidase 1 for the Treatment of Primary Hyperoxaluria Type 1.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder that leads to kidney and liver failure. PH1 is caused by a mutation in the alanine glyoxylate aminotransferase (AGXT) gene, which encodes a key metabolic enzyme that converts glyoxylate to glycine in the liver. Inability to metabolize glyoxylate leads to oxalate overproduction, yielding insoluble calcium oxalate crystals; accumulation of these crystals leads to progressive organ failure. Here, we used a novel, minimally disruptive genome-editing approach to disrupt the mechanism of action of hydroxyacid oxidase 1 (HAO1), an upstream enzyme in the glyoxylate metabolic pathway. Successful gene editing and disruption of the HAO1 gene is expected to increase levels of glycolate, a harmless intermediate of the glycine metabolic pathway, thereby preventing the formation of calcium oxalate crystals. We intravenously administered an adeno-associated virus (AAV) vector expressing the M1HAO1 meganuclease to both wild-type and Agxt-/- mice, a mouse model of PH1. We observed >30% editing of HAO1 in Agxt-/- mice, correlating with a dose-dependent increase in serum glycolate levels. At the highest dose tested, urine glycolate levels increased by 79%, with a concomitant 75% decrease in urine oxalate levels. We also evaluated in vivo targeting in rhesus macaques injected with AAV expressing two different versions of the HAO1 meganuclease. Dose-dependent editing of hepatic DNA and RNA was achieved, and serum glycolate levels changed in a manner consistent with successful liver editing; additionally, the treatment was well tolerated. Our results indicate that AAV-delivered meganucleases can effectively target HAO1 in mice and nonhuman primates to achieve high levels of HAO1 gene editing. Moreover, increased glycolate levels in serum indicate that this intervention significantly impacts the HAO1-mediated glycolate-to-glyoxylate pathway. These data suggest that this approach may represent an effective treatment for PH1.

Hyperoxaluria, Primary↗

RNA editing in plant mitochondria and chloroplasts.

In the mitochondria and chloroplasts of higher plants there is an RNA editing activity responsible for specific C-to-U conversions and for a few U-to-C conversions leading to RNA sequences different from the corresponding DNA sequences. RNA editing is a post-transcriptional process which essentially affects the transcripts of protein coding genes, but has also been found to modify non-coding transcribed regions, structural RNAs and intron sequences. RNA editing is essential for correct gene expression: proteins translated from edited transcripts are different from the ones deduced from the genes sequences and usually present higher similarity to the corresponding non-plant homologues. Initiation and stop codons can also be created by RNA editing. RNA editing has also been shown to be required for the stabilization of the secondary structure of introns and tRNAs. The biochemistry of RNA editing in plant organelles is still largely unknown. In mitochondria, recent experiments indicate that RNA editing may be a deamination process. A plastid transformation technique showed to be a powerful tool for the study of RNA editing. The biochemistry as well as the evolutionary features of RNA editing in both organelles are compared in order to identify common as well as organelle-specific components.

Amino Acid Sequence↗

CRM1 mediates the export of ADAR1 through a nuclear export signal within the Z-DNA binding domain.

RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.

Active Transport, Cell Nucleus↗

Identification of RNA editing sites in the SNP database.

The relationship between human inherited genomic variations and phenotypic differences has been the focus of much research effort in recent years. These studies benefit from millions of single-nucleotide polymorphism (SNP) records available in public databases, such as dbSNP. The importance of identifying false dbSNP records increases with the growing role played by SNPs in linkage analysis for disease traits. In particular, the emerging understanding of the abundance of DNA and RNA editing calls for a careful distinction between inherited SNPs and somatic DNA and RNA modifications. In order to demonstrate that some of the SNP database records are actually somatic modification, we focus on one type of these modifications, namely A-to-I RNA editing, and present evidence for hundreds of dbSNP records that are actually editing sites. We provide a list of 102 RNA editing sites previously annotated in dbSNP database as SNPs, and experimentally validate seven of these. Interestingly, we show how dbSNP can serve as a starting point to look for new editing sites. Our results, for this particular type of RNA editing, demonstrate the need for a careful analysis of SNP databases in light of the increasing recognition of the significance of somatic sequence modifications.

Base Sequence↗

Dyskinetoplastic Trypanosoma brucei contains functional editing complexes.

Mitochondrial pre-mRNAs undergo posttranscriptional RNA editing as directed by small guide RNAs (gRNAs) to produce functional mRNAs in kinetoplastid protozoa. The pre-mRNAs and gRNAs are encoded in the maxicircle and minicircle components, respectively, of the kinetoplastid mitochondrial DNA (kDNA), and editing is catalyzed by a multienzyme protein complex. Trypanosoma evansi AnTat3/3, which lacks maxicircles but retains a single class of minicircles, and a dyskinetoplastic mutant of Trypanosoma brucei EATRO164, which is devoid of kDNA, were both shown to retain genes and proteins for the editing complex. The proteins are present in complexes that immunoprecipitate and sediment indistinguishably from wild-type complexes. The complexes catalyze precleaved insertion and deletion editing as well as full-round deletion editing in vitro. Thus, mutants which lack the natural substrates for RNA editing and all or most gRNAs retain editing complexes that contain the four primary catalytic activities of editing and function in editing, at least in vitro. Therefore neither pre-mRNA nor gRNA is required to form functional RNA-editing complexes.

Animals↗

RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators.

APOBEC1 is the catalytic component of an RNA editing complex but shows homology to activation-induced cytidine deaminase (AID), a protein whose function is to potentiate diversification of immunoglobulin gene DNA. Here, we show that APOBEC1 and its homologs APOBEC3C and APOBEC3G exhibit potent DNA mutator activity in an E. coli assay. Indeed, like AID, these proteins appear to trigger DNA mutation through dC deamination. However, each protein exhibits a distinct local target sequence specificity. The results reveal the existence of a family of potential active dC/dG mutators, with possible implications for cancer.

APOBEC-1 Deaminase↗

RNA editing of apolipoprotein B mRNA. Sequence specificity determined by in vitro coupled transcription editing.

Apolipoprotein (apo) B-48 mRNA is produced by in vivo RNA editing which involves a C----U conversion of the first base of the codon CAA for Gln-2153, changing it to UAA, an in-frame stop codon. We have reproduced the editing reaction in vitro using nuclear extracts. Efficient RNA editing was demonstrated by using apoB mRNA segments as substrate or in a coupled transcription-editing reaction using apoB minigenes as template. ApoB minigenes were constructed by ligating the adenovirus major late promoter to a fragment of apoB-100 DNA containing the editing site and used for the transcription-editing reaction. We defined the sequence specificity of the editing reaction using site-specific single and multiple base mutants constructed by the polymerase chain reaction. Among 22 different mutant apoB-100 minigene constructs containing mutations in the bases immediately flanking the edited C-6666, 20 were edited in the coupled transcription-editing reaction. The results suggest a relatively lax sequence specificity for apoB mRNA editing. Our observation may have important implications for apoB-48 biogenesis as well as for the editing process as a general biologic regulatory mechanism.

Animals↗

From convention to innovation: the role of genetic modification and genome editing in Australian wheat breeding.

Wheat is the most cultivated crop worldwide, and Australia consistently ranks among the top wheat-exporting countries. Although modern technology has expanded the speed and accuracy of conventional breeding, progress is constrained by limited genetic diversity and linkage drag, with new wheat varieties often taking 8-12 years to reach the market. Biotech methods involving the transformation of foreign DNA into genomes [genetic modification (GM)], or editing of native DNA [genome editing (GEd)], provide novel opportunities to efficiently improve traits alongside conventional breeding. In 2020, the world's first GM drought-tolerant bread wheat (HB4) hit the market in Argentina. The USA recently approved HB4 wheat for commercial cultivation, and human consumption of HB4 wheat has been approved by nine countries, including Australia. Currently, 25 countries, Australia included, have deregulated GEd crops in some form, and many other countries have indicated that they will follow suit. As of March 2025, no GM or GEd wheat is commercially grown in Australia. The rate at which private industry integrates GM and GEd into wheat breeding programmes will depend on several factors, including the regulatory consistency governing GM and GEd crops within Australia and among international trading partners, the return on investments relative to deregulation costs including licensing, the level of acceptance amongst growers and consumers, and technical considerations including wheat's amenability to tissue culture. This review contextualizes GM and GEd applications in wheat, often drawing on examples from crop species where biotechnology has been more widely employed, and considers the key stakeholders that will shape the future of GM and GEd wheat in Australia.

GMO↗

Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.

Base Sequence↗

RNA-editing cytidine deaminase Apobec-1 is unable to induce somatic hypermutation in mammalian cells.

Antibody diversification by somatic hypermutation, gene conversion, and class switch recombination is completely dependent on activation-induced cytidine deaminase (AID). A recent report showing induction of DNA mutations in Escherichia coli by overexpression of AID, Apobec-1, and related members of the RNA-editing cytidine deaminase family suggested that they may directly modify deoxycytidine in DNA in mammalian cells (DNA-editing model). We therefore examined whether Apobec-1 bona fide RNA-editing enzyme could show somatic hypermutation and class switching activities in murine B lymphocytes and fibroblasts. Unlike AID, Apobec-1 was unable to induce somatic hypermutation or class switching. The results force a reevaluation of the physiological significance of the DNA deaminase activities of AID and Apobec-1 in E. coli and in vitro.

3T3 Cells↗

Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.

Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.

RNA Editing↗