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Results for “site-specific recombination”

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At least 19 recordsLinked to original sources
PubMed1979

A kappa-immunoglobulin gene is formed by site-specific recombination without further somatic mutation.

The active gene for a kappa light chain is formed by a somatic recombination event that joins one of several hundred variable region genes to one of a series of recombination sites (J-segments) encoded close to the kappa constant region gene. The nucleotide sequences of cloned germ line and somatically recombined genes define the precise organisation of these genetic segments and the site and nature of the recombination event that joined them. Apart from somatic recombination, no further alteration of ther germ line sequence has occurred. The J-segment is of special interest as it encodes signals for both DNA and RNA splicing and provides a means of generating further immunoglobulin gene diversity.

Animals↗
PubMed2026

Efficient scarless gene editing in Pichia pastoris via survival stress-based intramolecular homologous recombination.

To overcome low efficiency and/or genomic instability induced by DNA cleavage in current genome-editing approaches, a novel pop-in/pop-out-based editing system was developed for Pichia pastoris. An ingenious arrangement of components leads to a more efficient screening by permitting the only type of DNA recombination under defined pressure conditions, in terms of the overall efficiency of gene editing, the system virtually depends on the integration efficiency mediated by single-crossover recombination. It does not rely on exogenous recombinases or programmable nucleases such as Cas9, thereby avoiding nuclease induced double strand breaks and associated off target mutations or chromatin fatigue. This strategy preserves high editing efficiency with no modification to the host's inherent genetic properties. Relative to site-specific recombination methods, its dual MazF counterselection enables seamless editing, avoiding scar sequence-induced genomic instability. In this study, nearly 100% knockout efficiency and over 86.67% integration efficiency were achieved in the described experimental cases with this system, which provides a new gene-editing tool for synthetic biology in Pichia pastoris.

Efficient scarless editing↗
PubMed2026

Engineered genomic attachment sites for site-specific recombinases enable high-efficiency integration in plants and human cells.

Here we developed a DNA-centric strategy for optimizing site-specific recombination by rationally engineering chimeric attachment sites. The high-activity att variants enhance Bxb1-mediated integration efficiency in human cells and plants. Among these att variants, the engineered attB(V111) site achieved 51.9% integration efficiency in HEK293T cells (1.7-fold versus wild-type attB) and 35.6% in rice protoplasts (4.4-fold versus wild-type attB). When paired with an engineered single protein mutant in the Bxb1 catalytic domain, the optimized system achieved targeted integration efficiencies of 31% for a CD19 chimeric antigen receptor cassette and 25% for an ornithine transcarbamylase expression cassette in human cells. In rice, these engineered variants enabled integration of a 5.8 kb herbicide-resistance cassette at a targeted genomic locus, with stable integration detected in 24% of regenerated plants. Oxford Nanopore-based long-read sequencing of edited plants reveals complete and precise insertion with high specificity. Propagation of edited seedlings to T1 plants confirms heritable editing to future generations. This approach provides a safe, broadly applicable approach for recombinase-based genome editing.

Journal Article↗
PubMed2026

From feasibility to predictability: prime editing redefines precision breeding in plants.

Originally developed in mammalian systems as a genome editing strategy without double-strand breaks, prime editing (PE) has been adapted for precise genome modifications. However, its deployment revealed key limitations, including reduced efficiency, strong locus dependency, low germline transmission, and somatic chimerism. Consequently, diverse PE variants have emerged, resulting in fragmented landscape of architectures with context-dependent and inconsistent performance. This review consolidates these advances and outlines emerging design principles behind plant PE systems. It evaluates optimization strategies at multiple levels, discusses their applications in monocots and eudicots, and highlights persistent bottlenecks and future directions, including AI-guided protein engineering and improved delivery strategies. These advances position PE as a rapidly evolving platform toward enabling precision breeding in plants.

cis-regulatory engineering↗
PubMed1979

Sequences of five potential recombination sites encoded close to an immunoglobulin kappa constant region gene.

Immunoglobulin kappa chain gene formation involves site-specific somatic recombination between one of several hundred germ-line variable region genes and a joining site (or "J segment") encoded close to the constant region gene. We have cloned and determined the nucleotide sequence of major portions of the recombination region of the mouse kappa gene and discovered a series of five such J segments spread out along a segment of DNA 2.4 kilobases from the kappa constant region gene. These J segments encode the 13 COOH-terminal amino acids of the variable region, probably including amino acids involved in the antigen combining site and in heavy/light chain contacts. The J segments also display striking sequence homology to one another in both their coding and immediately flanking sequences. Major elements of a short palindrome--CAC(TA)GTG--are preserved adjacent to the recombination sites of both variable and J region genes and constitute inverted repeats at both ends of the sequences to be joined. These palindromes can be written as a hypothetical stem structure that draws variable and J regions together, providing a possible molecular basis for the DNA joining event. Four of the J segments that we have discovered encode amino acid sequences already found in myeloma proteins. By altering the frame of recombination, we can account for additional light chain amino acid sequences, suggesting that the V/J joining event might generate antibody diversity somatically both by using different combinations of variable and J region genes and by using alternative joining frames.

Animals↗
PubMed2026

A Tripartite AAV System with Engineered Lox Sites Enables Efficient Delivery of the EYS Gene for Retinal Gene Therapy.

Mutations in the Eyes Shut Homolog (EYS) gene are one of the leading causes of autosomal recessive retinitis pigmentosa, a progressive retinal degenerative disease for which no effective treatment currently exists. However, the large size of the EYS coding sequence (∼9.4 kb) exceeds the packaging limit of adeno-associated virus (AAV) vectors, posing a major barrier to gene replacement therapy. To address this challenge, we developed a tripartite AAV vector system that enables delivery and reconstitution of the full-length EYS gene using a Cre-lox-based unidirectional DNA recombination strategy, Uni-directional and Site-specific Transgene Assembly by Recombination (Uni-STAR). The system consists of three AAV constructs carrying discrete EYS segments flanked by engineered, noncompatible lox sites that drive ordered and unidirectional recombination in target cells. We validated this system in vitro by demonstrating successful reconstitution and expression of full-length EYS protein in HEK293T cells. In vivo, subretinal co-injection of the three AAV vectors into mouse eyes led to precise reconstitution and expression of full-length EYS protein in the retina. These findings establish the feasibility of using a tripartite AAV system to deliver the complete EYS gene and provide a foundation for future therapeutic development targeting EYS-associated retinal degenerations.

Dependovirus↗
PubMed2026

Site-Specific Measurement of Meiotic Crossing-Over Rate with Droplet Digital PCR.

Understanding the frequency and distribution of meiotic crossovers (COs) is critical for both fundamental studies on meiosis and for practical applications in plant breeding, where controlling recombination can accelerate crop improvement. Determining CO rates at specific genomic loci has traditionally relied on labor-intensive methods that require the production and genotyping of large progenies. Here, we present a high-throughput protocol for site-specific quantification of meiotic COs in maize using droplet digital PCR (ddPCR). The method is based on genotyping individual pollen nuclei from hybrid plants to detect recombinant and nonrecombinant alleles at defined chromosomal intervals. By distributing several thousands of pollen nuclei into nanoliter-sized droplets and performing PCR with allele-specific fluorescent probes, this method allows precise quantification of CO frequency with high sensitivity. The protocol provides detailed guidance for nuclei isolation, probe master mix preparation, droplet generation, and data interpretation. This method can be easily adapted for use in other plants.

Journal Article↗
PubMed2026

Cre-loaded integrase-defective lentiviral vectors for targeted cassette exchange in CHO cells.

Genome-modifying enzymes, such as recombinases and CRISPR-associated nucleases, enable targeted gene insertion when delivered transiently to minimize off-target effects. Precise genome engineering requires controlled enzyme activity, as well as efficient donor DNA transfer. Integrase-defective lentiviral vectors (IDLVs) provide a promising platform for transient episomal DNA transfer; however, their integration efficiency depends on complementary genome-targeting strategies. Here, we engineered Cre-loaded IDLVs (Cre-IDLVs) that co-package lentiviral vector genomes together with bioactive Cre recombinase. Cre was inserted into the Gag region of an integrase-defective gag-pol construct, allowing for efficient encapsidation and protease-mediated release during virion maturation without compromising the viral titer. The resulting particles carried donor cassettes flanked by heterospecific loxP sites. When applied to CHO founder cells harboring compatible genomic loxP landing pads, Cre-IDLVs efficiently mediated recombination-mediated cassette exchange, producing the highest number of G418-resistant colonies among the plasmid ratios tested. Genomic PCR and sequencing confirmed precise locus-specific insertion without detectable random integration in the analyzed clones. These findings establish Cre-IDLVs as a streamlined dual-delivery platform that couples transient recombinase activity with episomal donor DNA transfer. This hybrid lentiviral strategy provides a programmable approach for controlled and site-specific genome modification in mammalian cells.

Integrases↗
PubMed2010

Site-specific gene therapy for cardiovascular disease.

Gene therapy holds considerable promise for the treatment of cardiovascular disease and may provide novel therapeutic solutions for both genetic disorders and acquired pathophysiologies such as arteriosclerosis, heart failure and arrhythmias. Recombinant DNA technology and the sequencing of the human genome have made a plethora of candidate therapeutic genes available for cardiovascular diseases. However, progress in the field of gene therapy for cardiovascular disease has been modest; one of the key reasons for this limited progress is the lack of gene delivery systems for localizing gene therapy to specific sites to optimize transgene expression and efficacy. This review summarizes progress made toward the site-specific delivery of cardiovascular gene therapy and highlights selected promising novel approaches.

Animals↗
PubMed2026

Site-specific gene integration by recombinase-mediated cassette exchange in anhydrobiotic Pv11 cells.

Pv11 cells, derived from Polypedilum vanderplanki, uniquely tolerate complete desiccation (anhydrobiosis). Although a CRISPR/Cas9-based precise integration method (CRIS-PITCh) has been developed for Pv11 cells, a CRISPR-free strategy that fixes both the genomic locus and transgene copy number has not yet been established. Here, we implement recombinase-mediated cassette exchange (RMCE) in Pv11 cells and generate master cell lines that retain anhydrobiosis following genetic engineering. We first evaluated the activity of multiple site-specific recombinases in Pv11 cells using a transient two-plasmid reporter assay. Flp, Cre, and Bxb1 recombinases all excised a test cassette and activated a green fluorescent protein (GFP) reporter, whereas phiC31 integrase mediated recombination at the DNA sequence level but did not induce reporter expression under our construct configuration. To enable genomic RMCE, we inserted an FRT/FRT3-landing pad (LP) into a previously identified genomic safe-harbor locus using CRIS-PITCh and isolated clonal master cell lines by single-cell sorting. Using the established master line, Flp-based RMCE achieved site-specific cassette exchange at the LP, producing HaloTag fluorescence and drug resistance upon successful exchange. In addition, the expected post-exchange sequence was confirmed by sequencing. We further established an all-in-one RMCE vector combining the Flp recombinase and donor cassette on a single plasmid. Together, these results demonstrate locus-defined, single-copy transgene integration in anhydrobiotic Pv11 cells via RMCE and provide a standardized, CRISPR-free workflow for routine genetic manipulation in this unique cell line. This workflow facilitates both fundamental research and applied biotechnological applications using desiccation-tolerant cells.

Anhydrobiosis↗
PubMed2022

Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions.

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic ΔserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) ΔserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain ≥10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.

Humans↗
PubMed2025

Enzymatic Anti-Baldwin Ring-Closure Cascade for Fused Bicyclic Ether Formation.

Pyrenulic acids are cytotoxic polyketides isolated from the ascomycete Pyrenula sp. derived from Vietnamese lichen that are characterized by complex fused cyclic core structures. Genome sequencing, in silico sequence analysis, and RT-PCR studies identified the pyrenulic acid (pya) biosynthetic gene cluster. Based on a functional analysis of the enzymes by expression of each gene in a heterologous host using Aspergillus nidulans, we discovered two cytochrome P450s PyaJ and PyaG that effect epoxidation and hydroxylation of the alkyl chain terminal, respectively, and an α/β hydrolase PyaF that constructs a 6- and 7-membered fused bicyclic diether skeleton by catalyzing successive epoxide ring-opening 6-endo and 7-endo cyclization reactions. To elucidate the detailed mechanism of pyrenulic acid formation, we obtained PyaF as a recombinant enzyme and performed an in vitro experiment, which confirmed catalysis by PyaF of the cyclization reaction. In addition, we performed alignment analysis of PyaF with α/β hydrolases with known functions, as well as an in-depth computational study. In-depth computational analyses of the cyclization reaction pathways with density functional theory quantum mechanics and detailed characterization of PyaF by Chai-1-based protein structure modeling with molecular dynamics simulations and site-specific mutagenesis predicted the active amino acid residues of this serine α/β hydrolase to be an unusual catalytic serine tetrad involving Ser170, Asn342, Asp314, and His169, with Tyr255 and His284 acting as general bases to facilitate opening of the epoxides. Our study provides insight into how regioselectivity of enzymatic anti-Baldwin epoxide ring-opening cascades for the formation of a fused cyclic ether structure is controlled.

Cyclization↗
PubMed1978

Mechanism of integrating foreign DNA during transformation of Bacillus subtilis.

Genes encoding thymidylate synthetase from Bacillus subtilis bacteriophages were cloned in Escherichia coli. Chimeric plasmids pCD1 and pCD3 were constructed from site-specific endonuclease digests of bacteriophage phi3T DNA cloned in pMB9 in E. coli. Similar cloning techniques with bacteriophage beta22 DNA yielded chimeric plasmids pCD4, pCD5, and pCD6. Endonuclease digests of DNA from pCD1 and pCD3 propagated in E. coli or from DNA isolated from bacteriophage phi3T propagated in B. subtilis transformed B. subtilis from Thy- to Thy+. Intact DNA from bacteriophage beta22, endonuclease digests of beta22 DNA, and a chimeric plasmid (pCD5) composed only of the thybeta22 gene and pMB9 did not transform B. subtilis from Thy- to Thy+ even though pCD5 could transform Thy- E. coli to Thy+. However, if the thybeta22 fragment from pCD5 was introduced into another chimeric plasmid, pCD2, that contains a region of homology to the chromosome of B. subtilis in addition to pMB9, transformation of Thy- clones of B. subtilis was possible. Furthermore, Southern hybridization analyses of the digests of chromosomal DNA from the Thy+ transformants established that the entire chimeric plasmid was incorporated into the chromosome of B. subtilis. Treatment of these plasmids with site-specific endonucleases abolished transformation. These results indicated that the entire chimeric plasmid can be incorporated into the chromosome of B. subtilis by a Campbell-like model. Therefore, an additional mechanism for transformation exists whereby plasmids can be integrated if sufficient chromosomal homology is maintained.

Bacillus subtilis↗