Primer design remains a fundamental yet non-trivial step in modern molecular biology workflows. Although numerous tools are available, they are often fragmented across disparate applications, constrained by commercial licensing, or dependent on external servers, limiting workflow integration and compromising sequence confidentiality. To address this challenge, we developed PrimerWeaver (https://ignea.lab.mcgill.ca/primerweaver), a free browser-based tool that integrates primer design, quality control, and support for diverse cloning workflows within a single platform. PrimerWeaver enables overlap PCR, site-directed mutagenesis, multiplex PCR, restriction enzyme-based cloning (including Type II and Golden Gate assembly), and homology-directed methods such as Gibson assembly and Uracil-Specific Excision Reagent (USER) cloning. Primers were generated by optimizing the 3' annealing region for efficient amplification while appending workflow-specific 5' sequences according to the selected application. In silico and wet lab validation across diverse cloning workflows demonstrated consistent results relative to established molecular cloning software. All calculations are performed locally in the user's browser without sequence upload, preserving data confidentiality and supporting reproducible performance across systems. Overall, PrimerWeaver streamlines primer workflows for users ranging from undergraduate students to experienced researchers by integrating multiple PCR and cloning strategies into a single browser-based platform.

Highly repetitive DNA sequences remain difficult to synthesize, assemble, and verify, limiting their use in synthetic biology. Here, we report a modular plasmid framework for the scalable, sequence-defined assembly of repetitive DNA. As a model system, plasmids encoding repeats of the pentapeptide Gly-Val-Gly-Val-Pro (GVGVP)n were constructed to produce elastin-like polypeptides (ELPs) with temperature-dependent solubility. A synthetic DNA fragment encoding GVGVP17 was incorporated into a plasmid architecture that enables iterative repeat amplification through a Gibson-based digest-and-assemble workflow. Sequential HindIII and BamHI digestion followed by Gibson Assembly increased repeat number (2n-1 per cycle) while preserving plasmid architecture, yielding constructs up to GVGVP1025, as verified by whole-plasmid sequencing. A superfolder GFP was added to the GVGVP library with expression of up to 513 repeats and functional characterization up to 257 repeats in E. coli NEB 5-alpha cells. These results establish a generalizable strategy for constructing large repetitive DNA sequences and encoding programmable protein polymers.

Melon yellowing-associated virus (MYaV), a member of the genus Carlavirus (family Betaflexiviridae), is a positive-sense, single-stranded RNA virus with a poly(A) tail. It is thought to be one of the causative agents of severe yellowing disease in melon "Amarelão do meloeiro" (in Portuguese) in northeastern Brazil and is transmitted by the whitefly Bemisia tabaci. In the field, MYaV frequently occurs in mixed infections with the recombinant form of cucurbit aphid-borne yellows virus (CABYV), contributing to the disease complex of melon severe yellowing disease. Considering its impact, the inability of virus transmission by mechanical inoculation, and the lack of molecular tools to study this pathogen, we constructed a full-length infectious cDNA clone of MYaV using Gibson Assembly technology. The genome was amplified in two overlapping fragments spanning the 5' and 3' regions, cloned in the binary vector pJL89 and subsequently introduced to Agrobacterium tumefaciens strain GV3101. Agroinoculation with this construct revealed that the MYaV clone was infectious in melon, cucumber (Cucumis sativus), West Indian gherkin (Cucumis anguria), and watermelon (Citrullus lanatus). Notably, infections in watermelon and West Indian gherkin were asymptomatic, a finding of epidemiological relevance since these crops may act as unnoticed inoculum sources in the field for melon crops. In contrast, no infection was detected in squash (Cucurbita maxima) or zucchini (Cucurbita pepo). This infectious clone provides not only a valuable tool to advance studies on MYaV and virus-host interactions but also an essential resource for breeding programs aimed at developing resistant melon varieties.

Carbon dioxide (CO2) is a cost-effective, abundant, and renewable carbon source, but its utilization technologies face several issues. The reductive glycine pathway (RGP) is recognized as one of the most efficient one-carbon (C1) assimilation routes in nature, with its core component—the glycine cleavage system (GCS: GcvP, GcvH, GcvT, and GcvL)—playing an essential role in C1 metabolism. To develop efficient CO2 conversion and utilization pathways, we identified NhFtfL and AmFchA-MtdA with high catalytic efficiency through gene mining and constructed a four-plasmid co-expression system in E. coli BL21(DE3) using Gibson Assembly. This system integrated GcvP-GcvH, GcvT-GcvL, NhFtfL-AmFchA-MtdA, and RsPPK2, thereby reconstituting the complete RGP while enhancing ATP supply. The engineered strain functioned as an efficient whole-cell biocatalyst, achieving a glycine space–time productivity of 0.125 mmol/L/h via one-pot conversion of formate. Furthermore, we expanded the application scope by developing a whole-cell electrocatalysis system that directly synthesized glycine from CO2 and NH4Cl, achieving a glycine space–time productivity of 0.135 mmol/L/h. This study demonstrates the potential of the engineered RGP system for upgrading C1 resources and supports the transition toward carbon neutrality.

Marine microorganisms possess vast biosynthetic potential, yet most of their biosynthetic gene clusters (BGCs) remain transcriptionally silent under laboratory conditions. Genetic intractability has been a major barrier to activating these cryptic pathways. Here, we present RECC, an integrated Red/ET–CRISPR/Cas9 system that enables seamless, marker-free genome editing in marine bacteria. RECC couples Red/ET recombineering with CRISPR/Cas9-mediated cleavage, allowing the incorporation of homology arms and protospacers into a single construct through one-step Gibson assembly, thereby substantially simplifying the engineering process. Using Pseudoalteromonas flavipulchra DSM 14401 as a model, we employed RECC to replace the native promoter of a silent nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) hybrid gene cluster with a strong constitutive promoter. This targeted activation led to the production of a series of previously unknown cyclolipopeptides, designated flavipulchrins. Structural characterization and bioinformatic analysis revealed a plausible biosynthetic pathway for these metabolites. Collectively, RECC provides a robust and generalizable genome-editing platform that facilitates the systematic exploration of biosynthetic potential in genetically recalcitrant marine microorganisms.

This chapter describes the cloning strategy, expression, purification, and characterization of immunoglobulin G (IgG) and corresponding antibody fragments, namely fragment antigen-binding (Fab) and single-chain variable fragment (scFv). Theexamplesuse anti-HER2antibody, Trastuzumab, as a standard antibody. Thefirst method details a cloning strategy that enables the easy and quick reformatting of standard human IgG1 into antibody fragments using Gibson assembly. This approach generates two plasmids that bear either light chain or heavy chain genes, under the control of the CMV promoter. Thesecondmethod, and as an alternative design, presents a single vector approach carrying both light and heavy chain genes, where both genes are separated by an internal ribosome entry site (IRES). Finally, this chapter outlines the transient transfection process in Chinese hamster ovary (CHO) mammalian cells, followed by antibody purification and characterization steps for laboratory applications, highlighting key notes to achieve optimal results for IgGs, Fabs, and scFvs.

Microbial proteins hold great promise as sustainable alternatives for future protein sources, and oleaginous yeast Yarrowia lipolytica has emerged as a recognized platform for heterologous protein expression and secretion. N-terminal signal peptides (SPs) are crucial for directing proteins to the secretion pathway, which offers advantages in both academic and industrial protein production. Although some of the innate SPs of Y. lipolytica have been reported, there is a growing need to expand the genetic toolkit of SPs to support the increasing use of Y. lipolytica as a cell factory for overproduction of various secretory proteins. In this study, we employed an efficient evolutionary approach to rapidly evolve the innate SP XPR2-pre by leveraging Gibson assembly with two synthetic overlapping oligos containing high portion of degenerate nucleotides. Using Nanoluc (Nluc) luciferase as a robust reporter, we characterized the intracellular and extracellular enzymatic activity of 447 SP mutants and identified previously undescribed SPs exhibiting superior performance compared to XPR2-pre in Nluc luciferase secretion, with improvements of up to 2.91-fold of enzymatic activity in the supernatant. The generalizability of the top-performing SPs was evaluated using three additional heterologous enzymes (β-galactosidase, α-amylase, and PET hydrolase). Our results confirmed their versatility across different proteins with protein-specific efficiency. Additionally, based on our screening, we also evaluated the performance of different feature engineering strategies and machine learning models in the design and prediction of SP mutants. This study integrated rational design, directed evolution and machine learning to identify novel SPs, expanding the repertoire of signal peptides and benefiting secretory protein overexpression in Y. lipolytica.

Abstract BACKGROUND Glioblastoma (GBM) is a lethal, therapy-resistant brain tumor driven by glioma stem-like cells (GSCs). Despite progress in CAR-based immunotherapies in other cancers, their efficacy in GBM remains limited due to poor blood–brain barrier penetration and an immunosuppressive tumor microenvironment. In vivo immune engineering may overcome these obstacles. We propose a novel strategy to reprogram GSCs in situ into myeloid-like cells expressing a focused ultrasound (FUS)-inducible, HER2-specific CAR––enabling local, controllable fratricidal activity against HER2+GBM. FUS enables noninvasive, spatiotemporal regulation of CAR activity to minimize off-tumor toxicity. METHODS We constructed a heat/FUS-inducible HER2-CAR using SnapGene, PCR, and Gibson Assembly and confirmed its sequence by Sanger sequencing. CAR kinetics were validated in THP1 monocytes via heat shock and anti-CAR-Alexa Fluor 647 staining with flow cytometry. Separately, HER2+GSCs were transduced with the same CAR construct and verified for CAR expression. To reprogram GSCs, we co-expressed PU.1 and IKZF1––transcription factors first predicted computationally and then confirmed experimentally to drive GBM cells into dendritic and myeloid lineages––and enriched the converted cells by puromycin selection. We then quantified the conversion by flow cytometry for CD11b, CD45, CD80, CD86, MHC I, and MHC II surface myeloid-cell markers. RESULTS In THP1 cells, >50% of cells expressed the HER2-CAR 10 hours after heat induction (n=3). HER2 expression was confirmed in >70% of the cell population in three different GSCs. Reprogramming yielded 18% CD45⁺ and 33% CD86⁺ cells (n=3). Ongoing experiments are profiling additional myeloid markers across multiple GSC lines to assess platform robustness. CONCLUSION These data demonstrate the feasibility of reprogramming GSCs into controllable, CAR-expressing immune-like cells. This FUS-triggered in situ GBM fratricide approach lays the groundwork for a novel GBM immunotherapy.

Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide small RNAs designed to induce highly specific and efficient gene silencing in plants. Traditional approaches rely on the transgenic expression of ~1 kb TAS precursors, which limits their use in non-model species, under strict GMO regulations, and in size-constrained expression or delivery systems. This protocol describes a rapid workflow for the design, assembly, and delivery of syn-tasiRNAs derived from much shorter precursors, referred to as minimal precursors. The pipeline includes in silico design of highly specific syn-tasiRNA sequences, cloning of minimal precursors into plant expression or potato virus X (PVX)-based viral vectors through Golden Gate or Gibson assembly, and delivery to plants through Agrobacterium-mediated expression or by spraying crude extracts containing recombinant PVX expressing the minimal precursors. These methodologies make syn-tasiRNA-based tools more accessible and broadly applicable for plant research and biotechnology across diverse species and experimental contexts.Key features• Syn-tasiRNAs allow the simultaneous silencing of multiple genes with high specificity, as they are computationally designed to avoid off-target effects.• This protocol describes the design and obtention of syn-tasiRNAs for the simultaneous silencing of one or several endogenous genes in any plant species.• This protocol also describes a non-transgenic alternative for applying syn-tasiRNAs to plants using a viral vector to induce whole-plant gene silencing.• This protocol can also be applied to induce antiviral protection against pathogenic viruses, reducing viral mutational escapes when expressing multiple syn-tasiRNAs targeting different viral sites.

Abstract Background First detected in 1996 in geese in China, Highly Pathogenic Avian Influenza (HPAI) A(H5N1) virus cause severe illness with high mortality rates among infected poultry. Detection in human first occurred in 1997 during poultry outbreaks in Hong Kong and have since been detected in humans sporadically in more than 20 countries causing serious illness and death. In 2021, the HPAI A (H5N1) strain was detected in North America, primarily infecting wild birds and commercial poultry, though it has been spreading to wild terrestrial, marine, and even domestic animals. Human infections have also occurred both from exposure to infected birds and/or other animals, but also through unknown exposures. While no human-to-human transmission has occurred to date, the Center for Disease Control (CDC) considers HPAI A (H5N1) virus to be possible of causing severe disease in infected humans. Rapid testing of persons presenting with respiratory illness provides high value to health care providers, however the novel strain of HPAI A(H5N1) has been largely untested against diagnostic assays. This study evaluates laboratory detection of HPAI A(H5N1) using commercially available Nucleic Acid Amplification Tests (NAAT) with liquid-stable, qualitative proficiency samples containing whole genome H5N1 cDNA. Methods American Proficiency Institute, an independent proficiency testing provider, studied the recovery of HPAI A(H5N1) in simulated patient samples in a pilot event in 2025. Two blinded liquid samples (PIA-01 and PIA-02) supplied by Microbix Biosystems were mailed to 74 unique laboratories utilizing 15 different methodologies. Laboratories were instructed to process the sample as instructed by their Original Equipment Manufacturer’s (OEMs) Instructions for Use. Sample PIA-01 contained HPAI A (H5N1) and Sample PIA-02 contained H1N1. Sample PIA-01 was comprised of whole genome H5N1 cDNA template created using synthetic biology and Gibson assembly. The consensus sequence included was based on published data from the first human patients infected with H5N1 (clade 2.3.4.4b). Sequence was confirmed by NGS, quantified by ddPCR, and qualified for released by the Cepheid Gene Xpert SARS/Flu/RSV assay. Results Participants demonstrated they were able to accurately detect Influenza A, with only a limited number of laboratories being able to subtype to the level of H1N1 and H5N1. Conclusion Based on the results reported by participants for American Proficiency Institute’s pilot event, laboratories are able to detect HPAI A(H5N1) in NAAT assays as a positive result confirming Influenza A infection. While many methods do include limited subtyping A(H1) and A(H3), novel influenza A virus such as A(H5) are not commonly included in commercially available assays. Including subtypes is imperative to provide the necessary tracking in outbreaks.

We present a comprehensive web-based platform that integrates a suite of molecular biology tools tailored for PCR-based applications. Key features include custom multiplex tiling PCR panel design for amplicon sequencing, loop-mediated isothermal amplification (LAMP), allele-specific PCR genotyping assay development, Gibson assembly, oligonucleotide analysis and design, and the de novo identification, classification, and visualization of repetitive sequences. The platform supports a wide range of PCR primer design tasks, including standard, multiplex, reverse, long-range, quantitative fluorescence (TaqMan and MGB probe), and bisulfite PCR. In silico PCR analysis ensures primer and probe specificity across diverse applications such as gene discovery via homology analysis, molecular diagnostics, DNA profiling, and repeat sequence identification. The multiplex tiling PCR tool is optimized for both next-generation and third-generation sequencing technologies. LAMP primer design includes support for loop primers that target eight distinct regions of the DNA template. The allele-specific PCR module enables genotyping of single nucleotide polymorphisms, insertions/deletions, multi-nucleotide variants, and haplotypes at specific loci. Additionally, the platform facilitates genome-wide identification, masking, and clustering of repetitive elements, including interspersed repeats and low-complexity sequences such as simple sequence repeats and telomeres. All tools are freely accessible at https://primerdigital.com/tools/.

IntroductionSwine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging porcine enteric coronavirus that can cause diarrhea in piglets younger than 5 days of age. However, infection of pigs older than 5 days of age does not usually result in obvious clinical symptoms. This relative intrinsic safety in older animals prompted us to investigate the potential of SADS-CoV as a viral vector for porcine diarrhea virus vaccines.MethodsWe utilized Gibson assembly to clone the SADS-CoV sequence into an artificial bacterial chromosome (BAC) vector. Further manipulation was carried out by recombineering to generate four attenuated recombinant SADS-CoV strains expressing a PEDV protective antigen fused with peptides that target immune cells. Subsequently, the biological characteristics and immunogenic efficacy of these four recombinant strains were systematically assessed through in vitro cell models and in vivo animal challenge experiments.Results and discussionThe recombinant viruses exhibited a proliferation profile similar to that of the wild-type virus in Vero cells, maintained stable cytopathic effects, retained the exogenous sequences for up to 20 passages, and consistently expressed the PEDV antigen fusion protein. Immunizing pregnant sows with these recombinant viruses effectively enhanced both cellular and mucosal immune responses and provided significant clinical protection against PEDV to their offspring. This study not only generated a vaccine candidate for PEDV but also established a pipeline for using the SADS-CoV as a vector for vaccine development.

Efficient multi-gene expression in Escherichia coli is critical for advancing metabolic engineering and synthetic biology. However, existing strategies for combinatorial optimization remain labor-intensive and low-throughput. In addressing this challenge, a high-throughput platform was developed, encompassing the engineering of standardized genetic elements (promoters and 5' UTRs) with fluorescent reporters (e.g. eGFP, mCherry, TagBFP) to quantify expression variability. Libraries of single-, dual-, and tri-gene (dual-plasmid) constructs were assembled via Golden Gate, validated by IPTG induction, and applied to lycopene biosynthesis by replacing fluorescent genes with crtE, crtI, and crtB using Gibson assembly. The optimized tri-gene library was used to generate E. coli BL21(DE3) strains exhibiting variable levels of lycopene production, thereby demonstrating the platform's capacity to balance multi-gene pathways. Subsequent quantitative analysis by qPCR confirmed the uniformity of promoter-UTR combinations across the plasmid library. This modular platform, featuring reusable libraries and a dual-plasmid system, enables rapid exploration of multi-gene expression landscapes, offering a scalable tool for metabolic engineering and multi-enzyme co-expression.

The global spread of COVID-19 has underscored the urgent need for advanced tools to study emerging coronaviruses. Reverse genetics systems have become indispensable for dissecting viral gene functions, developing live-attenuated vaccine candidates, and identifying antiviral targets. In this study, we describe a robust and efficient reverse genetics platform for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The system is based on the assembly of a full-length infectious cDNA clone from seven overlapping fragments, each flanked by homologous sequences to facilitate seamless assembly using the Gibson assembly method. Individual cloning of each fragment into plasmids enables modular manipulation of the viral genome, allowing rapid site-directed mutagenesis by fragment exchange. Infectious recombinant virus was successfully recovered from the assembled cDNA, exhibiting uniform plaque morphology and genetic homogeneity compared to clinical isolates. Additionally, fluorescent reporter viruses were generated to enable real-time visualization of infection, and the effects of different mammalian promoters on viral rescue were evaluated. This reverse genetics platform enables efficient generation and manipulation of recombinant SARS-CoV-2, providing a valuable resource for virological research and the development of preventive and therapeutic antiviral measures.

DNA cloning methods are fundamental tools in molecular biology, synthetic biology, and genetic engineering that enable precise DNA manipulation for various scientific and biotechnological applications. This review systematically summarizes the major restriction-free overlapping sequence cloning (RFOSC) techniques currently used in synthetic biology and examines their development, efficiency, practicality, and specific applications. In vitro methods, including Gibson Assembly, Circular Polymerase Extension Cloning (CPEC), Polymerase Incomplete Primer Extension (PIPE), Overlap Extension Cloning (OEC), Uracil DNA Glycosylase-based Cloning (UDG-Cloning), and commercially available techniques such as In-Fusion, have been discussed alongside hybrid approaches such as Ligation-Independent Cloning (LIC), Sequence-Independent Cloning (SLIC), and T5 Exonuclease-Dependent Assembly (TEDA). Additionally, in vivo methods leveraging host recombination machinery, including Yeast Homologous Recombination (YHR), In Vivo Assembly (IVA), Transformation-Associated Recombination (TAR), and innovative approaches such as Phage Enzyme-Assisted Direct Assembly (PEDA), are critically evaluated. The review highlights that method selection should consider individual research projects' scale, complexity, and specific needs, noting that no single technique is universally optimal. Future trends suggest the increased integration of enzymatic efficiency, host versatility, and automation, broadening the accessibility and capabilities of DNA assembly technologies.

The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional characterization of genes across diverse biological processes. Current high-throughput methodologies for inter-vector DNA fragment transfer, however, necessitate PCR amplification of target sequences prior to cloning, rendering the generation of genome-scale plasmid collections technically demanding and time-intensive. By leveraging a CRISPRshuttle cassette, we developed a new high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which facilitates the transfer of many DNA fragments from donor plasmids sharing identical backbone sequences to a CRISPRshuttle-compatible vector without PCR amplification of the DNA fragments. Here, we present a protocol for CRISPRshuttle. This protocol involves two sequential test tube reactions prior to bacterial transformation. First, target DNA fragments are excised from donor plasmids by Cas9-mediated cleavage of their shared vector backbone sequence. Second, the excised DNA fragments are inserted into linearized CRISPRshuttle-compatible vectors through Gibson assembly. Our results demonstrate that the efficiency of CRISPRshuttle exceeds 94% and that two researchers can generate about 300 plasmids in 7 days using CRISPRshuttle. CRISPRshuttle facilitates efficient, adaptable, and cost-effective DNA fragment transfer between vectors, significantly streamlining genome-wide plasmid library generation.

Comprehensive SummaryAromatic polyketides (APKs) are renowned for their structural diversity and potent biological activities, making them valuable resources for drug discovery in antibiotics, anticancer agents, and antiparasitic therapies. Bipentaromycins, a unique class of dimeric APKs with a cyclic head‐to‐tail linkage, exhibit broad‐spectrum antibacterial activities. Acylation modifications in bipentaromycins C–F enhance their pharmacological properties, yet the responsible acyltransferase remains enigmatic. Herein, we present REGAIN, an innovative strategy combining restriction enzyme digestion, Gibson assembly, and in vivo Cre‐lox recombinatio n, enabling rapid and efficient cloning of biosynthetic gene clusters (BGCs). Using REGAIN, we successfully cloned a 40 kb bipentaromycin BGC (bpm). By integrating genetic experiments and computational modeling, we speculated BpmP as the acyltransferase responsible for regioselective acylation. This study establishes a robust platform for exploring novel bioactive molecules and advances the understanding of bipentaromycin biosynthesis.

The size and complexity of large viral genomes limit the technical possibilities for genome manipulations in fundamental research and medical or technological applications. State-of-the-art recombineering in bacteria has partially overcome this limit but remains a time-consuming and complex procedure requiring specialist expertise. Here, we describe a simplified and highly efficient in vitro protocol for unlimited and traceless manipulation applicable to large viral genomes from DNA viruses using a combination of CRISPR/Cas9 cleavage and in vitro DNA assembly. We successfully used the protocol to manipulate adenovirus genomes, showing that genome rescue from viruses, insertions, deletions, and mutagenesis can be performed in a simple overnight procedure in a standard laboratory setting without the need for advanced knowledge of molecular biology. Finally, we use our approach to demonstrate the de novo, multi-step construction of an adenovirus vector suitable for delivering very large transgenes for gene editing.IMPORTANCEThe 36 kb size of the adenoviral genome has long been a deterrent to the construction of adenoviral mutants by scientists wishing to study the virus itself or to construct adenoviral vectors for cell biology and gene therapy. Most previous techniques, such as recombineering and yeast gap repair, impress more by their elegance than by their ease. In this paper, we use Cas9 ribonucleoprotein particles (RNPs) to target cleavage to specific sites in an adenoviral plasmid, then repair the break by Gibson assembly. Gibson assembly with synthetic DNA fragments has transformed basic cloning. Combining it with Cas9 RNPs, which act like highly specific restriction enzymes, makes adenoviral mutagenesis as easy as traditional plasmid cloning. We have used the approach to modify multiple sites in the adenoviral genome, but it could be applied to any large DNA virus for which the genome can be cloned in a plasmid.

Comprehensive SummaryDNA synthesis and assembly technology is the fundamental enabling technology of synthetic biology, providing methods for humans to understand and modify organisms. Oligonucleotide chains and long DNA fragments synthesized through DNA synthesis and assembly technology are becoming increasingly widely used in fields such as biomedicine, energy, new materials, and information storage, with strong application prospects. This review provides a comprehensive and systematic introduction and exposition of current DNA synthesis and assembly technologies, discussing current challenges and future research prospects. Key ScientistsDNA synthesis and assembly technology is a cutting‐edge technology for human understanding and modification of living organisms, with wide applications in the fields of biomedicine, energy, new materials, and information storage. In 1962, the Bollum group first proposed that TDT enzyme could be used for the synthesis of DNA oligonucleotide chains. In 1987, the Carruthers group established column‐based oligo synthesis. In 1992, the Fodor group established microarray‐based oligo synthesis. In 1995, the Stemmer and Crameri groups established PCR based DNA assembly. In 2003, the Knight group proposed BioBricksTM. In 2007, the Elledge group was the first to report sequence and ligation‐independent cloning assembly. In 2008, three scientists Hamilton O. Smith, Clyde A. Hutchison III, and J Craig Venter co‐established transformation associated recombination technology. In the same year, the Marillonnet group established Gloden Gate technology. In 2009, the Gibson group established Gibson assembly technology. In 2012, the Zhang group established Red/Rec technology. In 2016, the Qin group established Cas9‐facilitated homologous recombination assembly. In the same year, the Dai group established Meiotic recombination‐mediated assembly. In 2017, the Bader group established switching auxotrophies progressively for integration. The Yuan group established yeast life cycle assembly in 2023 and established haploidization‐based DNA assembly and delivery in yeast in 2024. This review systematically introduces the latest research progress in DNA synthesis and assembly technology, providing a reference for subsequent research.

Abstract Background: Cancer remains one of the leading causes of mortality worldwide, with early detection pivotal to improving patient outcomes. Current diagnostic methods often lack the sensitivity and specificity to identify cancers at their earliest stages, especially for KRAS-driven cancers such as pancreatic cancer and non-small cell lung cancer (NSCLC). This challenge is further exacerbated for high-risk populations, where invasive biopsies, imaging, or endogenous biomarkers fail to meet clinical needs. There is a critical need for non-invasive, reliable, and continuous cancer detection methods capable of diagnosing tumors at nascent stages. To address this, we developed a mutation-mediated synthetic biomarker-based blood test specifically targeting early-stage cancers with KRAS G12 mutations. This study evaluates its performance in detecting KRAS-driven cancers using synthetic biomarkers and controls. Methods: A CRISPR-based gene editing system was designed to insert synthetic biomarker genes, such as Gaussia luciferase (GLuc), into cancer cells harboring KRAS G12 mutations. Using Gibson assembly, we constructed a donor plasmid carrying the GLuc gene alongside a plasmid encoding Cas9 VQR and sgRNA targeting the KRAS G12V locus. These plasmids were co-transfected into NCI-H727 human lung tumor cells. Single-cell sorting yielded 29 clonal lines with stable GLuc expression. Sequencing confirmed precise knock-in at the KRAS G12V target in 21 lines, with qPCR verifying exclusive insertion. GLuc secretion was measured via luciferase assays, and limits of detection (LOD) were assessed in vitro. To test biomarker detection in vivo, GLuc-expressing tumor cells were inoculated into SCID/NOD mice at varying numbers. Serum GLuc levels were measured using luciferase assays at defined time points. Results: In vitro, the LOD for GLuc-expressing cancer cells was 5,775 cells/mL. In vivo, SCID/NOD mice injected with GLuc-expressing tumor cells showed time-dependent increases in serum luminescence. Two weeks post-inoculation with 106 cells, serum luminescence in the experimental group was 8.24-fold higher than controls (8.24 ± 0.32), with a tumor lesion size of 29.86 ± 7.26 mm3. Conclusion: This mutation-mediated synthetic biomarker platform detected tumor lesions as small as millimeter-scale in vivo with high sensitivity and specificity after a single injection. Its non-invasive, scalable design offers the potential to transform early detection and monitoring of KRAS-driven cancers, addressing critical unmet needs for high-risk populations. Citation Format: Shengyue Piao, Miller Harris, Kevin McHugh. Early mutation-mediated detection of cancers via biomarker production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB156.