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PubMed2003

Regulation of human apolipoprotein B gene expression at multiple levels.

Apolipoprotein B is a large, amphipathic protein that plays a central role in lipoprotein metabolism. Because its overproduction and deficiency leads to metabolic and pathologic disorders, much effort has been paid to investigate the mechanisms of how its homeostasis is achieved. Earlier and recent studies have showed that apoB gene locus might reside in different chromatin domains in the hepatic and intestinal cells, and two sets of very distinct regulatory elements operate to control its transcription. Posttranscriptional modification of apoB mRNA is performed by a multicomponent enzyme complex, several possible pathways regulate the editing efficiency. Understanding of the mechanism responsible for apoB mRNA editing will provide the basis for C-to-U editing in gene therapy. In addition to apoB mRNA abundance and stability, its translation can be also regulated at the steps of elongation. The translocation of apoB into the ER is an important and complicated process that is less understood. Successful transport and correct folding of apoB may lead to its final secretion, otherwise subject to intracellular degradation, which is accomplished by proteasomal and nonproteasomal pathways at multiple levels and may differ among cell types.

Animals↗
PubMed2025

Fast Fission Yeast Genome Editing by CRISPR/Cas9 Using Gap Repair and Fluoride Selection.

We present a protocol to perform CRISPR/Cas9-mediated genome editing in the fission yeast Schizosaccharomyces pombe that does not require cloning and uses the fluoride exporter channel Fex1 as the selection marker. Transformation is typically carried out on the same day of PCR primer arrival and successfully edited strains are selected 5 days after transformation. We expect the adoption of this protocol to further accelerate the throughput of genome editing in S. pombe.

Schizosaccharomyces↗
PubMed1998

Rearrangement of lambda light chain genes in mature B cells in vitro and in vivo. Function of reexpressed recombination-activating gene (RAG) products.

V(D)J (V, variable; D, diversity; J, joining) combination of immunoglobulin (Ig) genes established in premature B cells has been thought to be conserved throughout differentiation at mature stages. However, germinal center (GC) B cells have been shown to reexpress recombination-activating gene (RAG)-1 and RAG-2 proteins in immunized mice. Here, we present several lines of evidence indicating that RAG proteins thus induced are functional as the V(D)J recombinase. DNA excision product reflecting Vlambda1 to Jlambda1 rearrangement was generated in parallel with the expression of RAG genes in mature mouse B cells that were activated in vitro with LPS and IL-4. Similar lambda chain gene rearrangement was observed in the draining lymph node of immunized mice. Further, B cells that underwent lambda gene rearrangement were shown by in situ PCR to be localized within GCs. Thus, secondary rearrangement of Ig genes (receptor editing) can occur in mature B cells.

Animals↗
PubMed2025

Engineering recombination machinery facilitates the construction of yeast cell factories.

Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.

Metabolic Engineering↗
PubMed2026

Enhancing CRISPR-Cas12a base editing in plants with LbCas12a variants and introns.

Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools for precise genome editing in plants. Conventionally, such base editors are built upon the CRISPR-Cas9 systems where Cas9 nickases are used. To expand the base editing scope and minimize off-target effects, base editors derived from the CRISPR-Cas12a systems are desired. However, the use of deactivated Cas12a (dCas12a) in such base editors constrains the editing activity, preventing the wide use of Cas12a base editors for plant research and trait development. In this study, we demonstrate the use of an ABE based on the efficient LbCas12a-RRV variant to introduce herbicide-resistant mutations in OsACCase in rice. To improve Cas12a CBEs and ABEs, we inserted introns into the coding sequence of dLbCas12a-RRV. This intron-containing Cas12a-CBE shows substantial improvement in editing efficiency in rice, compared to the intron-less counterparts. By contrast, the improvement of ABE with the intron-containing dLbCas12a-RRV is very limited, partly due to the already high baseline editing efficiency of the intron-less dLbCas12a-RRV ABE. Testing of these base editors in poplar shows elevated C-to-T base editing by dLbCas12a-RRV-intron-CBE. For A-to-G editing, ABEs built upon dLbCas12a-RV and dLbCas12a-RRV variants showed significant improvement over ABEs derived from wild-type LbCas12a and the ttLbCas12a variant. The addition of introns to dLbCas12a-RRV does not further improve the base editing efficiency. With whole genome sequencing in rice, we evaluated genome editing specificities with these improved Cas12a base editors. Our analyses show that both intron-containing Cas12a CBE and ABE barely introduce guide RNA-dependent off-target mutations. However, they can generate guide RNA-independent off-target mutations, which are likely attributed to the high enzymatic activities of the deaminases. Collectively, our study demonstrates the successful use of a Cas12a base editor for trait development and reports improved Cas12a CBEs and ABEs for precise base editing in plants.

Oryza↗
PubMed1990

RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing.

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to-U transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited atp9 mRNA is translated.

Adenosine Triphosphatases↗
PubMed2026

Genome Editing for Glycogen Storage Diseases.

Gene therapy has been developed for several glycogen storage diseases and has advanced into clinical trials. However, the limitations of these gene therapies with regard to stability following treatment early in life have led to the development of genome editing. Early results for genome editing in both glycogen storage disease type Ia and Pompe disease have demonstrated promising efficacy, and proof-of-concept studies as well as a clinical trial are underway. These studies will determine whether genome editing fulfills its promise with regard to stably treating glycogen storage diseases early in life.

Humans↗
PubMed1994

HOVERGEN: a database of homologous vertebrate genes.

Comparison of homologous genes is a major step for many studies related to genome structure, function or evolution. Similarity search programs easily find genes homologous to a given sequence. However, only very tedious manual procedures allow the retrieval of all sets of homologous genes sequenced for a given set of species. Moreover, this search often generates errors due to the complexity of data to be managed simultaneously: phylogenetic trees, alignments, taxonomy, sequences and related information. HOVERGEN helps to solve these problems by integrating all this information. HOVERGEN corresponds to GenBank sequences from all vertebrate species, with some data corrected, clarified, or completed, notably to address the problem of redundancy. Coding sequences have been classified in gene families. Protein multiple alignments and phylogenetic trees have been calculated for each family. Sequences and related information have been structured in an ACNUC database which permits complex selections. A graphical interface has been developed to visualize and edit trees. Genes are displayed in color, according to their taxonomy. Users have directly access to all information attached to sequences and to multiple alignments simply by clicking on genes. This graphical tool gives thus a rapid and simple access to all data necessary to interpret homology relationships between genes. HOVERGEN allows the user to easily select sets of homologous vertebrate genes, and thus is particularly useful for comparative sequence analysis, or molecular evolution studies.

Animals↗
PubMed2026

CRISPR-Cas9‑based lipid nanocarriers for advanced therapy of urinary bladder cancer.

Bladder cancer (BCa) exhibits significant genetic and phenotypic variability. This variability suggests that various tumor subtypes could be influenced by several biomarkers and signaling pathways, which presents a problem for monotherapy strategies. Despite the initial effectiveness of traditional therapies, BCa's high rates of progression and recurrence, and the eventual development of drug resistance in many patients, continue to be major concerns. Because of the potential to transform the genetic modifications linked to the disease, genome editing using CRISPR/Cas9 has become a transformative tool in medicine with noteworthy potential for BCa therapy. Although the CRISPR/Cas9 technology is incredibly effective at transforming the field of genome editing, its instability and cellular impermeability pose significant challenges to its delivery. To increase efficient delivery of CRISPR/Cas9, nanovectors may be investigated. Significant promise exists for improving the therapeutic potential of CRISPR-Cas9 technology and addressing complex cancer therapy difficulties because of the rapid development of nanotechnology-based delivery systems. Relevant articles were searched in Google Scholar, Scopus, and Web of Science covering studies published between 2007 and 2026. Along with the impact of lipid-based nanoparticles on their safe transport to cancer cells, this review emphasizes the significance of the CRISPR/Cas9 genome editing system in the treatment of BCa.

CRISPR–Cas9↗
PubMed2026

Integrating genomics, multi-omics, CRISPR and speed breeding for stress-resilient vegetable legume improvement.

Vegetable legumes are nutritionally and ecologically important crops. However, their genetic improvement has not kept pace with the increasing challenges posed by climate change due to the polygenic nature of stress tolerance, narrow genetic diversity, and the persistent gap between molecular discoveries and field-level cultivar development. Although recent reviews have examined individual genomic tools or specific stress responses, a comprehensive synthesis integrating genomics-assisted breeding, multi-omics technologies, genome editing, and speed breeding within a unified crop improvement framework has been lacking. This review addresses that gap by critically evaluating how these complementary approaches can accelerate the development of stress-resilient vegetable legumes, including pea, common bean, cowpea, faba bean, cluster bean, yard-long bean, and hyacinth bean. This review synthesizes advances in QTL mapping, genome-wide association studies, transcriptomics, metabolomics, and CRISPR-based functional genomics that have identified key regulators and pathways underlying resistance to major biotic and abiotic stresses. Rather than considering these technologies independently, the review emphasizes their convergence into a systems-level breeding framework integrating genomic discovery, functional validation, predictive breeding, and accelerated generation advancement to improve breeding efficiency. Speed breeding, enabling up to seven to eight generations annually under optimized controlled-environment experimental conditions in cowpea, is discussed as a complementary strategy with genomic selection and genome editing. The review further identifies major translational bottlenecks, including transformation recalcitrance, limited genomic resources for underutilized vegetable legumes, inadequate multi-environment validation, and fragmented omics integration, and presents an integrated systems-breeding framework to bridge the gap between gene discovery and cultivar development.

Fabaceae↗
PubMed2025

Genome editing research initiatives and regulatory landscape of genome edited crops in India.

Food and nutritional security are the top priorities in Indian agriculture. Exponential population growth coupled with climate change effects has become a serious challenge for sustainable agriculture. Genome editing has revolutionized the agricultural sector because of its ability to create precise, stable and predictable modifications in the genome and therefore, offers great opportunities for crop improvement in India. However, for harvesting the real benefits of this technology in agriculture sector, there is a strong need of creating awareness among the end users and development of suitable policies for regularization of genome edited products. Many regulatory agencies around the world have been modernizing their regulatory approaches to be more risk proportionate and to reflect a more science-based approach. In this article, recent research initiatives and developments undertaken by different Indian institutes/organizations for the genetic improvement of agricultural and horticultural crops via genome editing technologies are summarized. Furthermore, to benefit from this potential technology in our country, regulatory policies must be clear, science-based and proportionate. Therefore, in the present review, the regulatory policies related to the genome editing of crop products in India are discussed in detail. This review will sensitize researchers and stakeholders to the application of genome editing techniques in crop improvement and various biosafety committees involved in the development and regulation of genome edited crops.

Crops, Agricultural↗
PubMed2004

Thermodynamic modeling of donor splice site recognition in pre-mRNA.

When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 small nuclear RNA with the donor ( 5(') ) splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our "finding with binding" method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

Base Pairing↗
PubMed2024

Global quantification of off-target activity by base editors.

Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.

Gene Editing↗
PubMed2025

Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations. However, existing models have a limited ability to create multiple targeted edits, hindering our understanding of complex genetic interactions. Here we generate transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), which robustly generates compound genotypes, including diverse cancers driven by inactivation of trios of tumour suppressor genes or an oncogenic translocation. We integrate these modular CRISPR RNA (crRNA) arrays with clonal barcoding to quantify the size and number of tumours with each array, as well as the impact of varying the guide number and position within a four-guide array. Finally, we generate tumours with inactivation of all combinations of nine tumour suppressor genes and find that the fitness of triple-knockout genotypes is largely explainable by one- and two-gene effects. These Cas12a alleles will enable further rapid creation of disease models and high-throughput investigation of coincident genomic alterations in vivo.

Animals↗
PubMed2026

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

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

Animals↗
PubMed2026

Programmable enzymes for targeted gene insertion.

Genome editing technologies have advanced from nuclease-based reagents that generate programmed DNA double-strand breaks, which can cause deleterious effects, to next-generation reagents that perform controlled DNA modification through double-strand break-independent mechanisms, such as base editing and prime editing. Although these approaches enable precise small-scale sequence changes, methods for programmable insertion of large DNA cargos have been limited. The ability to write entire genes or large regions into the genome could transform the treatment of genetically heterogeneous disorders, for which numerous pathogenic variants underlie a common disease and mutation-specific editing strategies are impractical. Recent advances in computational genome mining have accelerated the discovery of naturally occurring enzymes with novel biochemical and functional properties, including recombinases and transposases capable of large-scale modifications. Moreover, directed evolution, rational engineering and expanded homologue discovery are enabling the repurposing and optimization of these systems for genome engineering. Here we review recent technology development efforts that harness diverse enzymes for kilobase-scale genome engineering, with a particular focus on CRISPR-associated transposase systems.

Journal Article↗
PubMed2001

New approaches to lymphoma diagnosis.

Recent years have brought an explosion of new diagnostic tools to the pathology of lymphomas, which have permitted more precise disease definition and recognition of factors that can predict prognosis and response to treatment. These new methods exploit both the biological features of normal lymphocytes as they progress through differentiation pathways and the genetic abnormalities that characterize malignant transformation. These features can be assessed in individual tumors with techniques that detect proteins (immunophenotyping), messenger RNA (in-situ hybridization), or changes in DNA [Southern blot, PCR, fluorescence in-situ hybridization (FISH), and gene sequencing]. Recently, the novel technology of "gene chips" or DNA microarrays has greatly enhanced the efficiency of analyzing expression of many genes simultaneously at the RNA level. Understanding the relationship of lymphoid neoplasms to their normal counterparts and the genetic events that lead to malignant transformation in lymphoid cells are essential for physicians caring for patients with lymphoma, since these are the basis of modern classification, diagnosis, and prognosis prediction. Although microarray technology is not ready for prime time in the daily diagnosis of lymphoma, practitioners should understand its potential and limitations. The vast majority of lymphoid neoplasms worldwide are derived from B lymphocytes at various stages of differentiation. The review by Harald Stein and colleagues present the events of normal B-cell differentiation that are relevant to understanding the biology of B-cell neoplasia. These include antigen receptor [immunoglobulin (Ig)] gene rearrangement, somatic mutations of the Ig variable region genes, receptor editing, Ig heavy chain class switch, and differential expression of a variety of adhesion molecules and receptor proteins as the cell progresses from a precursor B cell to a mature plasma cell. Most lymphoid neoplasms have genetic abnormalities, many of which appear to occur during the gene rearrangements and mutations that characterize normal B-cell differentiation. Dr. Riccardo Dalla Favera reviews the mechanisms of these translocations and other abnormalities, and their consequences for lymphocyte biology. The association of specific abnormalities with individual lymphomas is reviewed. Dr. Wing C. Chan reviews the technology and applications of DNA microarray analysis, its promises and pitfalls, and what it has already told us about the biology of lymphomas. Finally, what does this all mean? The applications, both current and future, of these discoveries to the diagnosis and treatment of patients with lymphoma are discussed by Dr. Nancy Lee Harris.

Cell Differentiation↗