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PubMed2025

A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.

Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.

Bacteria↗
PubMed2025

Systematic Optimization Enables Near-Perfect In Vitro Transformation Efficiencies for Spirodela polyrhiza (Greater Duckweed).

The in vitro transformation of plants, or the delivery of foreign genetic material that is incorporated into their genomes, represents a powerful tool both for elucidating genotype-phenotype relationships and for generating plant cultivars which have desirable traits for agriculture and/or biotechnological applications. However, outside of a few model species, the processes involved in transformation are often inefficient and can take months to perform for many plant species, with several bottlenecks occurring at the different stages of calli induction, genetic transfection, and plant regeneration. While duckweeds - aquatic monocots whose species include some of the smallest and fastest-growing flowering plants on the planet - have distinguished themselves with several emerging biotechnological applications, they too are the subject of conflicting reports regarding their transformation potential and ability to be genetically manipulated. Here, we synthesized and optimized the protocols for in vitro transformation of duckweed Spirodela polyrhiza (Greater Duckweed) from start-to-finish: achieving >90% - 100% efficiencies for each of calli induction; transient and stable genetic transformation; visual marker-free selection of transformants; and regeneration of genetically modified plants with stable transgene expression for over 100 generations - and which in S. polyrhiza can be achieved over the course of weeks instead of months. The integrated, streamlined approaches for all stages of in vitro transformation overcome many bottlenecks and can help to pave the way for high-throughput functional genomics studies and synthetic biology applications in this biotechnologically-important species.

CRISPR/Cas9↗
PubMed2025

Cr3a, a candidate gene conferring fruit cracking resistance, was fine-mapped in an introgression line of Solanum lycopersicum L.

In the cultivation and production of tomato (Solanum lycopersicum L.), fruit cracking is a prevalent and detrimental issue that significantly impacts the esthetic quality and commercial value of the fruit. The complexity of the trait has resulted in a slow advancement in research aimed at identifying genes that influence tomato fruit cracking and the underlying regulatory mechanisms. In this study, a sub-introgression population for tomato crack-resistant fruit has been constructed from the cross between S. lycopersicum 1052 and Solanum pennellii LA0716, followed by 11 generations of selfing. Utilizing specifically designed InDel markers, the tomato crack-resistant gene, Cr3a, was fine-mapped, cloned, and its functionality was confirmed through transgenic and gene-knockout approaches. The precise localization of Cr3a was delineated to a 30 kb genomic region on chromosome 3, corresponding to the gene Sopen03g034650 in S. pennellii and Solyc03g115660.3 in the Heinz1706 variety. An integrated transcriptomic and metabolomic analysis of fruits with and without the Cr3a gene was finally conducted to elucidate the intricate regulatory mechanisms associated with Cr3a. The findings revealed a molecular regulatory network for tomato fruit crack resistance, characterized by 7 key metabolites, 13 pivotal genes, and 4 critical pathways: the phenylpropanoid biosynthesis pathway, the phenylalanine, tyrosine, and tryptophan biosynthesis pathway, the linolenic acid metabolism pathway, and the cysteine and methionine metabolism pathway. In summary, this research provides novel insights into the molecular underpinnings of tomato fruit crack resistance and holds substantial promise for accelerating the molecular breeding of tomatoes with enhanced fruit crack resistance.

Solanum lycopersicum↗
PubMed2006

Overcoming resistance to interferon-induced apoptosis of renal carcinoma and melanoma cells by DNA demethylation.

Epigenetic editing of gene expression by aberrant methylation of DNA may help tumor cells escape attack from the innate and acquired immune systems. Resistance to antiproliferative effects and apoptosis induction by interferons (IFNs) was postulated to result from silencing of IFN response genes by promoter hypermethylation. Treatment of human ACHN renal cell carcinoma (RCC) and A375 melanoma cells with the DNA demethylating nucleoside analog 5-AZA-2'-deoxycytidine (5-AZA-dC) synergistically augmented antiproliferative effects of IFN- alpha (alpha) 2 and IFN-beta (beta). Either 5-AZA-dC or an antisense to DNA methyltransferase 1 (DNMT1) overcame resistance to apoptosis induction by IFNs with up to 85% apoptotic cells resulting from the combinations. No similar potentiation occurred in normal kidney epithelial cells. IFN response genes were augmented more than 10 times in expression by 5-AZA-dC. Demethylation by 5-AZA-dC of the promoter of the prototypic, apoptosis-associated IFN response gene XAF1 was confirmed by methylation-specific polymerase chain reaction. siRNA to XAF1 inhibited IFN-induced apoptosis; conversely, overexpression of XAF1 overcame resistance to apoptosis induction by IFN-beta. As occurred with apoptosis-resistant melanoma cells in vitro, tumor growth inhibition in the nude mouse of human A375 melanoma xenografts resulted from treatment with 5-AZA-dC in combination with IFN-beta, an effect not resulting from either single agent. The importance of epigenetic remodeling of expression of immune-modifying genes in tumor cells was further suggested by identifying reactivation of the cancer-testis antigens MAGE and RAGE in ACHN cells after DNMT1 depletion. Thus, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumor-associated antigens.

Adaptor Proteins, Signal Transducing↗
PubMed2024

Non-replicative phage particles delivering CRISPR-Cas9 to target major blaCTX-M variants.

Cluster regularly interspaced short palindromic repeats and CRISPR associated protein 9 (CRISPR-Cas9) is a promising tool for antimicrobial re-sensitization by inactivating antimicrobial resistance (AMR) genes of bacteria. Here, we programmed CRISPR-Cas9 with common spacers to target predominant blaCTX-M variants in group 1 and group 9 and their promoter in an Escherichia coli model. The CRISPR-Cas9 was delivered by non-replicative phagemid particles from a two-step process, including insertion of spacer in CRISPR and construction of phagemid vector. Spacers targeting blaCTX-M promoters and internal sequences of blaCTX-M group 1 (blaCTX-M-15 and -55) and group 9 (blaCTX-M-14, -27, -65, and -90) were cloned into pCRISPR and phagemid pRC319 for spacer evaluation and phagemid particle production. Re-sensitization and plasmid clearance were mediated by the spacers targeting internal sequences of each group, resulting in 3 log10 to 4 log10 reduction of the ratio of resistant cells, but not by those targeting the promoters. The CRISPR-Cas9 delivered by modified ΦRC319 particles were capable of re-sensitizing E. coli K-12 carrying either blaCTX-M group 1 or group 9 in a dose-dependent manner from 0.1 to 100 multiplicity of infection (MOI). In conclusion, CRISPR-Cas9 system programmed with well-designed spacers targeting multiple variants of AMR gene along with a phage-based delivery system could eliminate the widespread blaCTX-M genes for efficacy restoration of available third-generation cephalosporins by reversal of resistance in bacteria.

CRISPR-Cas Systems↗
PubMed2023

Turning up the heat on essential E. coli genes.

Temperature-sensitive (TS) alleles create tunable thermoswitches to deplete essential cellular activities and are used to dissect gene function. In their recent study, Link and colleagues (Schramm et al 2023) use a CRISPR-based approach to systematically create TS alleles across essential genes in E. coli.

Escherichia coli↗
PubMed2004

[The application of chloroplast genome transformation in higher plant].

The chloroplast genome transformation of higher plant has become the research hotspot of plant genetic engineering with several advantages over nuclear genetic engineering, and it has become into a powerful approach for both basic and applied research. This paper presents a brief summary for the principle and methods of chloroplast genome transformation in higher plant. The particular emphasis was placed on its application in the basic and applied research. These applications include the studies of rearrangement of Rubisco and the structure, transcription, translation and RNA editing of genes in chloroplast; the producing of antibodies, vaccines, poly-beta-hydroxybutyrate and bio-elastic protein using chloroplast as bioreactor; the generating of transgenic plants with resistance to insect, disease, herbicide and drought; and the decreasing the gene flow of transgenic plants.

Chloroplasts↗
PubMed1999

RNA editing of a Drosophila sodium channel gene.

Extensive analysis of cDNAs from the para locus in D. melanogaster reveals posttranscriptional modifications indicative of adenosine-to-inosine RNA editing. Most of these edits occur in highly conserved regions of the Na+ channel, and they occur in distant relatives of D. melanogaster as well. Sequence comparison between species has identified putative cis-acting elements important for each RNA editing site. Double-stranded RNA secondary structures with striking similarity to known RNA editing sites were generated based on these data. In addition, the RNA editing sites appear to be developmentally regulated. We have cloned a potential RNA editase, DRED, with a high degree of homology to the mammalian RED1,2 genes. The DRED locus itself is highly regulated by transcription from alternative promoters and alternative splicings.

Adenosine Deaminase↗
PubMed2026

Duchenne muscular dystrophy: from gene to gene-ius therapies.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional dystrophin, leading to progressive muscle degeneration, loss of ambulation, respiratory failure, cardiomyopathy, and premature mortality. Despite advances in multidisciplinary supportive care, DMD remains an incurable disease associated with substantial physical, psychosocial, and economic burdens. The monogenic nature of DMD and its well-defined molecular pathogenesis have made it a prime target for the development of precision therapies aimed at restoring dystrophin expression or modifying disease progression. This review provides an overview of the genetic and molecular mechanisms underlying DMD, summarizes its clinical manifestations and natural history, and discusses current standards of care. It further examines recent advances in disease-modifying therapeutic strategies, including exon-skipping antisense oligonucleotides, nonsense mutation readthrough agents, adeno-associated virus (AAV)-mediated micro-dystrophin gene replacement, and emerging genome-editing technologies such as CRISPR/Cas9. The review also highlights the limitations of existing treatments, including mutation specificity, variable efficacy, immune-related challenges, and uncertainties regarding long-term durability and safety. Finally, it considers future directions in therapeutic development, emphasizing the need for combination approaches, improved delivery systems, and next-generation gene-editing platforms to achieve more effective and lasting clinical outcomes. Collectively, these advances represent a paradigm shift in DMD management and offer renewed hope for improving survival and quality of life for affected individuals.

Humans↗
PubMed1998

Demonstration of mRNA editing and localization of guide RNA genes in kinetoplast-mitochondria of the plant trypanosomatid Phytomonas serpens.

Maxicircle molecules of kDNA in several isolates of Phytomonas were detected by hybridization with the 12S rRNA gene probe from Leishmania tarentolae. The estimated size of maxicircles is isolate-specific and varies from 27 to 36 kb. Fully edited and polyadenylated mRNA for kinetoplast-encoded ribosomal protein S12 (RPS12) was found in the steady-state kinetoplast RNA isolated from Phytomonas serpens strain 1G. Two minicircles (1.45 kb) from this strain were also sequenced. Each minicircle contains two 120 bp conserved regions positioned 180 degrees apart, a region enriched with G and T bases and a variable region. One minicircle encodes a gRNA for the first block of editing of RPSl2 mRNA, and the other encodes a gRNA with unknown function. A gRNA gene for the second block of RPSl2 was found on a minicircle sequenced previously. On each minicircle, a gRNA gene is located in the variable region in a similar position and orientation with respect to the conserved regions.

Amino Acid Sequence↗
PubMed1993

In vitro transcription and replication of the mumps virus genome.

By the use of lysolecithin-permealized extracts from mumps virus-infected HeLa cells, we have developed an in vitro system, which not only directed the synthesis of mumps virus mRNAs but also supported replication of the genomic RNA. Furthermore, upon transcription of the P gene, both faithful and edited copies of the P gene were detected by RNase mapping with a riboprobe. Thus this system seems to promote biochemical analyses of underlying mechanisms operative in mumps virus gene expression and replication, including RNA editing.

Cell-Free System↗
PubMed1996

Apolipoprotein B mRNA editing protein: a tool for dissecting lipoprotein metabolism and a potential therapeutic gene for hypercholesterolemia.

Apolipoprotein (apo) B mRNA editing protein is an essential catalytic component of the apoB mRNA editing enzyme complex. Its cDNA has been cloned recently from rats and humans. In the presence of other proteins of the editing enzyme complex, it will deaminate nucleotide 6666 in apoB mRNA, a cytidine residue, converting it to uridine. The end result of this reaction is the production of apoB-48 in place of apoB-100. The editing protein exists as a homodimer. It imparts editing activity to HepG2, a human hepatoma cell line, causing these cells to start producing apoB-48 in addition to apoB-100. Therefore, it can be used as a therapeutic agent to reduce apoB-100 production. Preliminary experiments in our laboratory indicate that somatic gene transfer of the editing protein is highly effective in lowering plasma low density lipoproteins.

APOBEC-1 Deaminase↗
PubMed1991

A termination codon is created by RNA editing in the petunia atp9 transcript.

Analysis of the cDNA of the atp9-1 gene transcript from petunia mitochondria has revealed that ten C residues of the gene sequence are edited into U in the mRNA. Seven of these edits result in amino acid changes and one introduces a stop codon before the end of the open reading frame predicted from the gene sequence. The resulting protein is better conserved when compared to the same protein in other organisms. Comparison of the edited petunia sequence with other plant mitochondrial atp9 gene sequences idicates variation in the number and positions of edits required to obtain the same amino acids in ATP9 polypeptides of higher plants.

Amino Acid Sequence↗
PubMed2026

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

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

Humans↗
PubMed1995

Immunoglobulin heavy chain gene replacement: a mechanism of receptor editing.

We have generated a site-directed transgenic (sd-tg) mouse model in which the JH locus has been replaced with a rearranged VDJ coding for the heavy chain of an anti-DNA antibody. In these mice, B cells expressing the anti-dsDNA specificity are negatively regulated. We observe a novel mechanism for B cell tolerance, receptor editing at the heavy chain locus. In most sd-tg B cells, the inserted anti-DNA VH gene has been replaced by the upstream endogenous VH, or DH, or both genes through recombination with the heptamer embedded at the 3' end of most VH genes. Three types of recombination events have been identified. VH-to-VDJ, DH-to-VDJ, and VH-to-DH-VDJ. Analysis of the junctional sequences revealed features of classical V(D)J rearrangement, namely N sequence addition and nucleotide deletion. A conserved nonamer was found 12 bp upstream of the embedded heptamer. This nonamer may represent a novel recombination signal sequence used for VH editing. The sd-tg model thus provides direct evidence for secondary rearrangement at VH-D-JH. This process may play a role in tolerance by editing autoreactive receptors and may also serve to diversify the VH repertoire.

Amino Acid Sequence↗
PubMed1994

Chylomicron retention disease: exclusion of apolipoprotein B gene defects and detection of mRNA editing in an affected family.

Chylomicron retention disease (CRD) is a rare autosomal recessive disorder characterized by the absence of post-prandial chylomicrons and apolipoprotein (apo) B-48 in sera from affected individuals. Apo B-100 is synthesized, and apo B-100-containing lipoproteins are present in sera. A crucial difference between the synthesis and secretion of apo B-containing lipoproteins from the liver and gut in man is the generation of apo B-48 by editing of apo B mRNA in the gut to create a premature stop-translation codon. In this study the hypothesis that CRD may represent an absence of editing of apo B mRNA in the gut was investigated. Two affected sisters were identified as having low cholesterol levels and an absence of post-prandial chylomicronemia. Segregation analysis in the family showed that the apo B locus is not the site of the defect. Using reverse transcription-polymerase chain reaction (RT-PCR), duodenal biopsy-mRNA from the affected sisters was isolated and analyzed. The apo B editing site was amplified after cDNA synthesis, and the products analyzed by the primer extension assay. The results show that editing of apo B mRNA is normal in patients with CRD. The data provides strong confirmation that the primary defect in CRD is not in the synthesis, or editing of apo B mRNA in the gut. More likely, the disease arises from a defect in a gene crucial to the assembly and/or secretion of the chylomicron particle.

Apolipoprotein B-48↗
PubMed1990

A stuttering model for paramyxovirus P mRNA editing.

Paramyxovirus P genes are transcribed into two mRNAs which differ from each other by either one (measles and Sendai virus) or two (SV5 and mumps virus) G insertions, and which code for either the P or V proteins. The G insertions always occur within a short run of Gs, and a stuttering mechanism for the insertions has been suggested in which the viral polymerase reiteratively copies a template C residue during mRNA synthesis. Support for this mechanism was obtained by varying the reaction conditions during Sendai virus mRNA synthesis in vitro. A stuttering model is proposed which accounts for how the ratio of inserted to uninserted mRNAs is controlled, and why some paramyxoviruses insert one G and others two Gs when insertions occur.

Base Sequence↗
PubMed1997

Tissue-specific and differential editing of the two ycf3 editing sites in maize plastids.

The hypothetical chloroplast open reading frame 3 (ycf3) of maize, consisting of three exons and two group II introns, contains two editing sites. Both of these sites were investigated with respect to the extent of editing in various tissues and different developmental stages. Northern blot analyses show nearly identical transcript patterns of ycf3 in all tissues investigated. In leaf plastids, both editing sites are completely edited, independent of light conditions and developmental stage. In non-leaf plastids, however, one editing site of ycf3 is only partially edited in unspliced transcripts and in one type of partially spliced transcripts. In different developmental stages of the same tissue, on the other hand, no differences in editing efficiency were found. These results indicate that, in partially spliced transcripts, different editing sites of one and the same gene can be edited with different efficiencies in a tissue-specific manner.

Open Reading Frames↗