Assembly Of Vectors Research Articles

4G cloning: rapid gene assembly for expression of multisubunit protein complexes in diverse hosts.

Multisubunit protein complexes are central to many cellular processes, and studying their activities and structures in vitro requires reconstitution via recombinant expression and purification. Obtaining targets at sufficient purity and scale typically involves screening several protein variants and expression hosts. Existing cloning strategies enable co-expression but are often time-consuming, labor-intensive, and host-specific, or involve error-prone steps. We present a novel vector set and assembly strategy to overcome these limitations, enabling expression construct generation for multisubunit complexes in a single step. This modular system can be extended to additional hosts or include new tags. We demonstrate its utility by constructing expression vectors for structural maintenance of chromosomes complexes in various hosts, streamlining workflows, and improving productivity.

Blood biodistribution and vector shedding of valoctocogene roxaparvovec in people with severe hemophilia A

AbstractFollowing systemically administered adeno-associated virus gene therapy, vector particles are widely distributed, raising concerns about horizontal or germline vector transmission. Characterization of biodistribution and kinetics of vector DNA in body fluids can address these concerns and provide insights into vector behavior in accessible samples. We investigated biodistribution and vector shedding profile of valoctocogene roxaparvovec in men with severe hemophilia A enrolled in the phase 3 GENEr8-1 trial. Participants (n = 134) received a single 6 × 1013 vector genome (vg)/kg infusion and were assessed over 3 years. Vector DNA was measured using 4 different assays. Total vector DNA was evaluated in blood, saliva, stool, semen, and urine by quantitative polymerase chain reaction (qPCR). Encapsidated vector DNA was measured in plasma and semen with immunocapture-based qPCR. Contiguity of vgs and assembly of inverted terminal repeat fusions were measured in whole blood and peripheral blood mononuclear cells (PBMCs) using multicolor digital PCR. Median peak vector DNA levels observed 1 to 8 days after dosing were highest in blood, followed by saliva, semen, stool, and urine. Concentrations declined steadily. Encapsidated vector DNA cleared faster than total vector DNA, achieving clearance by ≤12 weeks in plasma and semen. Predominant vector genome forms transitioned from noncontiguous to full-length over time in whole blood and PBMCs, indicating formation of stable circularized episomes within nucleated cells. The replication-incompetent nature of valoctocogene roxaparvovec, coupled with steady clearance of total and encapsidated vector DNA from shedding matrices, indicates transmission risk is low. This trial was registered at www.ClinicalTrials.gov as #NCT03370913.

Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis.

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography-tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an attractive host for the expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilizes type IIS restriction enzymes to facilitate an efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of 16 well-characterized SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion promoting signals are validated by using five different proteins of interest. Comparison of intracellular and secreted protein levels reveals the optimal secretion promoting sequence for each individual protein. Large, protein of interest-specific variations in secretion efficiency are observed. SP sequences are also used with the five surface display anchors, and the combination of SP and anchor protein proves critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs and TFPs and anchor proteins for optimal secretion and/or surface display of a given protein of interest in S. cerevisiae.

Modular vector assembly enables rapid assessment of emerging CRISPR technologies

The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.

Assembly of Baculovirus Vectors for Multiplexed Prime Editing.

Efficient genome editing by using CRISPR technologies requires simultaneous and efficient delivery of multiple genetically encoded components to mammalian cells. Amongst all editing approaches, prime editing (PE) has the unique potential to perform seamless genome rewriting, in the absence of DNA double-strand breaks (DSBs). The cargo capacity required for efficient PE delivery to mammalian cells stands at odd with the limited packaging capacity of traditional viral delivery vectors. By contrast, baculovirus (BV) has a large synthetic DNA capacity and can efficiently transduce mammalian cells. Here we describe a protocol for the assembly of baculovirus vectors for multiplexed prime editing in mammalian cells.

Efficient Gene-Editing in Human Pluripotent Stem Cells Through Simplified Assembly of Adeno-Associated Viral (AAV) Donor Templates.

Gene-edited human pluripotent stem cells provide attractive model systems to functionally interrogate the role of specific genetic variants in relevant cell types. However, the need to isolate and screen edited clones often remains a bottleneck, in particular when recombination rates are sub-optimal. Here, we present a protocol for flexible gene editing combining Cas9 ribonucleoprotein with donor templates delivered by adeno-associated virus (AAV) vectors to yield high rates of homologous recombination. To streamline the workflow, we designed a modular system for one-step assembly of targeting vectors based on Golden Gate cloning and developed a rapid protocol for small-scale isolation of AAV virions of serotype DJ. High homology-directed repair (HDR) rates in human pluripotent stem cells (hPSCs), ~70% in ACTB and ~30% in LMNB1, were achieved using this approach, also with short (300 bp) homology arms. The modular design of donor templates is flexible and allows for the generation of conditional and/or complex alleles. This protocol thus provides a flexible and efficient strategy workflow to rapidly generate gene-edited hPSC lines. Key features • Versatile approach combining AAV-DJ donors and CRISPR ribonucleoproteins, providing an efficient method for long and short edits, insertions, and deletions in human pluripotent stem cells. • One-step cloning method for rapid generation of customized AAV donor plasmids. • Simplified AAV purification pipeline for ready-to-infect virion preparations.

High-efficiency computation for electromagnetic forming process: An explicit-implicit GPU approach

Low computational efficiency is one of the crucial challenges in electromagnetic forming process simulation. However, the huge gap in switching from the electromagnetic field to mechanics hinders the development of high-efficiency parallel coupling. A graphics processing unit (GPU)-based explicit-implicit coupling method is proposed to address the high-computational cost in simulations of the electromagnetic forming process, in which the large deformation (explicit) and electromagnetic field (implicit) are intimately coupled. The electromagnetic-mechanical coupling is achieved by constructing five parallel algorithms, which include the parallel operations of vector-vector, matrix-matrix, matrix-vector, vector assembly, and matrix-free assembly parallel preconditioned conjugate gradient algorithm. The high-efficiency GPU parallel computing is realized through the special data storage structure and parallel strategies. The explicit-implicit GPU method is validated against the experimental results, and several examples are presented for simulations of the electromagnetic forming process to illustrate both the accuracy and high-efficiency parallel acceleration performance of the present program.

Streamlined assembly of cloning and genome editing vectors for genus Clostridium

Reported herein is a new set of vectors designed to streamline molecular cloning and genome editing by exploiting modern cloning methods. The new vectors build on the existing pMTL8000 vectors that have been a staple of Clostridium research for more than a decade. The introduction of two pairs of type IIS restriction sites flanking an insulated multiple cloning site in both a cloning vector and a CRISPR-Cas9 gene editing vector enables plasmid construction in a "one-pot" reaction, avoiding the more laborious steps of conventional cloning. A synthetic lacZα expression cassette introduced between the cloning sites enables visual detection of background colonies. In addition, distinct selection markers on each vector permit selection of the desired clones according to antibiotic resistance. An example of strain development using the new vectors is demonstrated.

Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.

Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.

Easy method for six-fragment Golden Gate Assembly of modular vectors.

Efficient cloning techniques are a requirement for synthetic biology. This study provides a simplified cloning method based on Golden Gate Assembly that can be used for rapid vector construction. The building of multiple expression vectors with customizable modules is achieved in a timely manner with minimal hands-on time by removing unnecessary steps in the workflow. The authors constructed a total of 21 mammalian episomal expression vectors and conducted a fluorescence expression comparison for different regulatory region combinations post-transfection in HEK293T and HEPG2 cells. Screening revealed that using the EF-1α promoter in combination with the bovine growth hormone polyadenylation sequence seemed to perform best in the types of cells tested compared with other variants.

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana.

Vinblastine is a chemotherapy agent produced by the plant Catharanthus roseus in small quantities. Currently, vinblastine is sourced by isolation or semisynthesis. Nicotiana benthamiana is a plant heterologous host that can be used for reconstitution of biosynthetic pathways as an alternative natural product sourcing strategy. Recently, the biosynthesis of the late-stage vinblastine precursors precondylocarpine acetate, catharanthine, and tabersonine have been fully elucidated. However, the large number of enzymes involved in the pathway and the unstable nature of intermediates make the reconstitution of late-stage vinblastine precursor biosynthesis challenging. We used the N.benthamiana chassis and a state-of-art modular vector assembly to optimize the six biosynthetic steps leading to production of precondylocarpine acetate from the central intermediate strictosidine (∼2.7 mg per 1 g frozen tissue). After selecting the optimal regulatory element combination, we constructed four transcriptional unit assemblies and tested their efficiency. Finally, we successfully reconstituted the biosynthetic steps leading to production of catharanthine and tabersonine.

An Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants.

Transgenic technology can transfer favorable traits regardless of reproductive isolation and is an important method in plant synthetic biology and genetic improvement. Complex metabolic pathway modification and pyramiding breeding strategies often require the introduction of multiple genes at once, but the current vector assembly systems for constructing multigene expression cassettes are not completely satisfactory. In this study, a new in vitro gene stacking system, GuanNan Stacking (GNS), was developed. Through the introduction of Type IIS restriction enzyme-mediated Golden Gate cloning, GNS allows the modular, standardized assembly of target gene expression cassettes. Because of the introduction of Gateway recombination, GNS facilitates the cloning of superlarge transgene expression cassettes, allows multiple expression cassettes to be efficiently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-mediated marker deletion mechanism. The linked dual positive-negative marker selection strategy ensures the efficient acquisition of target recombinant plasmids without prokaryotic selection markers in the T-DNA region. The host-independent negative selection marker combined with the TAC backbone ensures the cloning and transfer of large T-DNAs (>100 kb). Using the GNS system, we constructed a binary vector containing five foreign gene expression cassettes and obtained transgenic rice carrying the target traits, proving that the method developed in this research is a powerful tool for plant metabolic engineering and compound trait transgenic breeding.

GeneTargeter: Automated In Silico Design for Genome Editing in the Malaria Parasite, Plasmodium falciparum.

Functional characterization of the multitude of poorly described proteins in the human malarial pathogen, Plasmodium falciparum, requires tools to enable genome-scale perturbation studies. Here, we present GeneTargeter (genetargeter.mit.edu), a software tool for automating the design of homology-directed repair donor vectors to achieve gene knockouts, conditional knockdowns, and epitope tagging of P. falciparum genes. We demonstrate GeneTargeter-facilitated genome-scale design of six different types of knockout and conditional knockdown constructs for the P. falciparum genome and validate the computational design process experimentally with successful donor vector assembly and transfection. The software's modular nature accommodates arbitrary destination vectors and allows customizable designs that extend the genome manipulation outcomes attainable in Plasmodium and other organisms.

Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering.

With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate the rapid design and assembly of vectors. However, such systems approach a technical bottleneck when a library of bespoke vectors is required. Utilizing the flexibility and robustness of the Extensible Mammalian Modular Assembly (EMMA) toolkit, we present an automated workflow for the library-scale design, assembly, and verification of mammalian expression vectors. Vector design is simplified using our EMMA computer-aided design tool (EMMA-CAD), while the precision and speed of acoustic droplet ejection technology are applied in vector assembly. Our pipeline facilitates significant reductions in both reagent usage and researcher hands-on time compared with manual assembly, as shown by system Q-metrics. To demonstrate automated EMMA performance, we compiled a library of 48 distinct plasmid vectors encoding either CRISPR interference or activation modalities. Characterization of the workflow parameters shows that high assembly efficiency is maintained across vectors of various sizes and design complexities. Our system also performs strongly compared with manual assembly efficiency benchmarks. Alongside our automated pipeline, we present a straightforward strategy for integrating gRNA and Cas modules into the EMMA platform, enabling the design and manufacture of valuable genome editing resources.

EMMA-CAD: Design Automation for Synthetic Mammalian Constructs.

Computational design tools are the cornerstone of synthetic biology and have underpinned its rapid development over the past two decades. As the field has matured, the scale of biological investigation has expanded dramatically, and researchers often must rely on computational tools to operate in the high-throughput investigational space. This is especially apparent in the modular design of DNA expression circuits, where complexity is accumulated rapidly. Alongside our automated pipeline for the high-throughput construction of Extensible Modular Mammalian Assembly (EMMA) expression vectors, we recognized the need for an integrated software solution for EMMA vector design. Here we present EMMA-CAD (https://emma.cailab.org), a powerful web-based computer-aided design tool for the rapid design of bespoke mammalian expression vectors. EMMA-CAD features a variety of functionalities, including a user-friendly design interface, automated connector selection underpinned by rigorous computer optimization algorithms, customization of part libraries, and personalized design spaces. Capable of translating vector assembly designs into human- and machine-readable protocols for vector construction, EMMA-CAD integrates seamlessly into our automated EMMA pipeline, hence completing an end-to-end design to production workflow.

Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus nidulans as a Heterologous Host.

Fungal natural products encompass an important source of pharmaceutically relevant molecules. Heterologous expression of biosynthetic pathways in chassis strains enables the discovery of new secondary metabolites and characterization of pathway enzymes. In our laboratory, biosynthetic genes in a clustered pathway have been refactored in engineered heterologous hosts such as Aspergillus nidulans. Here we describe the assembly of heterologous expression vectors, transformation into A. nidulans, and detection of new compounds in the transformant strains.

Cloning and sequencing of the promoter and terminator regions of the rbcL gene from Gunnera perpensa L.

Primers were designed based on the chloroplast genome sequence from the cotton Gossypium barbadense L. Specificrestriction sites were included in the primers so as to facilitate the process of vector assembly for future chloroplast transformation. Polymerase chain reaction (PCR) was applied to amplify the promoter and terminator regions of the rbcLgene from the chloroplast DNA (cpDNA) of G. perpensa. These promoter and terminator regions of the rbcL gene were around 0.25 and 0.5 kilobase pair (kb) in length, respectively. The amplicons were purified and cloned into pGEM®-T Easy Vector System I, then transformed into DH5α strain of E. coli bacteria. Transformed bacteria were propagated and recombinant plasmids were extracted and sequenced. The resulted sequences were aligned and compared with the sequenced promoter and terminator of rbcL from cotton as the reference plant. The promoter region of rbcL gene from G. perpensa showed 81.5% identity with that of the cotton plant, while the terminator region of rbcL gene expressed 69.9% identity with that of cotton plant. The core sequences at -35 and -10 of the promoter from G. perpensa scored 54.44% of thedefault core sequences

CT5, a subtle in vitro DNA assembling method based on the combination of FnCas12a and T5 exonuclease.

To develop a new DNA assembly method based on FnCas12a and T5 exonuclease. We developed a method named as FnCas12a and T5 exonuclease (CT5) cloning system. FnCas12a performs site-directed cleavage to the target DNA fragments, and T5 exonuclease generates 20-30 nt single-stranded region at each end of the DNA fragments for homologous recombination-mediated DNA assembly. CT5 was applied to multi-fragment assembly and DNA cloning of large vectors (> 10kb). The efficiencies were approximately 91.4% and 97%, respectively. In addition, CT5 cloning is also utilized for the "walking" of DNA elements, which enables subtle modification of the relative distances of DNA elements in plasmids. The CT5 method was a precise and exquisite DNA operating system and provided an ideal platform for the study of gene functions, genetic engineering and synthetic biology.

Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants.

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.