Golden Gate Cloning Research Articles

A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9

BackgroundThe yeast Komagataella phaffii (formerly known as Pichia pastoris) has been widely used for functional expression of recombinant proteins, including plant and animal food proteins. CRISPR/Cas9 genome editing systems can be used for insertion of heterologous genes without the use of selection markers. The study aimed to create a convenient markerless knock-in method for integrating expression cassettes into the chromosome of K. phaffii using CRISPR/Cas9 technology. The approach was based on the hierarchical, modular, Golden Gate assembly employing the GoldenPiCS toolkit. Furthermore, the aim was to evaluate the system’s efficiency and suitability for producing secreted recombinant food proteins.ResultsThree Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids containing homology regions for chromosomal integration via homology-directed repair. The integration efficiency of an enhanced green fluorescent protein (eGFP) expression cassette was assessed at three genomic loci (04576, PFK1, and ROX1). The 04576 locus showed the highest integration efficiency, while ROX1 had the highest transformation efficiency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fluorescence. Furthermore, the system’s applicability for producing recombinant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the first report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin.ConclusionsThe adapted GoldenPiCS toolkit combined with CRISPR/Cas9 technology enabled efficient and precise genome integration in K. phaffii. This approach holds promise for expanding the production of high-value recombinant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system’s efficiency and versatility.

Golden Gate cloning enables efficient concatemer construction for biophysical analysis of heterozygous potassium channel variants from patients with epilepsy.

Golden Gate cloning enables efficient concatemer construction for biophysical analysis of heterozygous potassium channel variants from patients with epilepsy.

RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides.

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ (R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. This result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides, providing a streamlined method for addressing genetic engineering challenges in the yeast.

Modularization of the type II secretion gene cluster from Xanthomonas euvesicatoria facilitates the identification of a structurally conserved XpsCLM assembly platform complex.

Many bacterial pathogens depend on a type II secretion (T2S) system to secrete virulence factors from the periplasm into the extracellular milieu. T2S systems consist of an outer membrane secretin channel, a periplasmic pseudopilus and an inner membrane-associated assembly platform including a cytoplasmic ATPase. The components of T2S systems are often conserved in different bacterial species, however, the architecture of the assembly platform is largely unknown. Here, we analysed predicted assembly platform components of the Xps-T2S system from the plant-pathogenic bacterium Xanthomonas euvesicatoria. To facilitate these studies, we generated a modular xps-T2S gene cluster by Golden Gate assembly of single promoter and gene fragments. The modular design allowed the efficient deletion and replacement of T2S genes and the insertion of reporter fusions. Mutant approaches as well as interaction and crosslinking studies showed that the predicted assembly platform components XpsC, XpsL and XpsM form a trimeric complex which is essential for T2S and associates with the cytoplasmic ATPase XpsE and the secretin XpsD. Structural modeling revealed a similar trimeric architecture of XpsCLM homologs from Pseudomonas, Vibrio and Klebsiella species, despite overall low amino acid sequence similarities. In X. euvesicatoria, crosslinking and fluorescence microscopy studies showed that the formation of the XpsCLM complex is independent of the secretin and vice versa, suggesting that the assembly of the T2S system is a dynamic process which involves the association of preformed subcomplexes.

Modular Cloning of Multigene Vectors for the Baculovirus System and Yeast.

Modular Cloning of Multigene Vectors for the Baculovirus System and Yeast.

A rapid and efficient strategy for combinatorial repression of multiple genes in Escherichia coli

BackgroundThe regulation of multiple gene expression is pivotal for metabolic engineering. Although CRISPR interference (CRISPRi) has been extensively utilized for multi-gene regulation, the construction of numerous single-guide RNA (sgRNA) expression plasmids for combinatorial regulation remains a significant challenge.ResultsIn this study, we developed a combinatorial repression system for multiple genes by optimizing the expression of multi-sgRNA with various inducible promoters in Escherichia coli. We designed a modified Golden Gate Assembly method to rapidly construct the sgRNA expression plasmid p3gRNA-LTA. By optimizing both the promoter and the sgRNA handle sequence, we substantially mitigated undesired repression caused by the leaky expression of sgRNA. This method facilitates the rapid assessment of the effects of various inhibitory combinations on three genes by simply adding different inducers. Using the biosynthesis of N-acetylneuraminic acid (NeuAc) as an example, we found that the optimal combinatorial inhibition of the pta, ptsI, and pykA genes resulted in a 2.4-fold increase in NeuAc yield compared to the control.ConclusionWe anticipate that our combinatorial repression system will greatly simplify the regulation of multiple genes and facilitate the fine-tuning of metabolic flow in the engineered strains.

Optimizing in vitro Transcribed CRISPR-Cas9 Single-Guide RNA Libraries for Improved Uniformity and Affordability.

We describe a scalable and cost-effective sgRNA synthesis workflow that reduces costs by over 70% through the use of large pools of microarray-derived oligos encoding unique sgRNA spacers. These sub-pool oligos are assembled into full-length dsDNA templates via Golden Gate Assembly before in vitro transcription with T7 RNA polymerase. RNA-seq analysis reveals severe biases in spacer representation, with some spacers being highly overrepresented while others are completely absent. Consistent with previous studies, we identify guanine-rich sequences within the first four nucleotides of the spacer, immediately downstream of the T7 promoter, as the primary driver of this bias. To address this issue, we introduced a guanine tetramer upstream of all spacers, which reduced bias by an average of 19% in sgRNA libraries containing 389 spacers. However, this modification also increased the presence of high-molecular-weight RNA species after transcription. We also tested two alternative bias-reduction strategies: compartmentalizing spacers within emulsions and optimizing DNA input and reaction volumes. Both methods independently reduced bias in 2,626-plex sgRNA libraries, though to a lesser extent than the guanine tetramer approach. These advancements enhance both the affordability and uniformity of sgRNA libraries, with broad implications for improving CRISPR-Cas9 screens and optimizing guide RNA design for other CRISPR and nuclease systems.

Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping.

Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli.

Cloning methods are fundamental to synthetic biology research. The capability to generate custom DNA constructs exhibiting predictable protein expression levels is crucial to the engineering of biology. Golden Gate cloning, a modular cloning (MoClo) technique, enables rapid and reliable one-pot assembly of genetic parts. In this study, we expand on the existing MoClo toolkits by constructing and characterizing compatible low- (p15A) and medium-copy (pBR322) destination vectors. Together with existing high-copy vectors, these backbones enable a protein expression range covering a 500-fold difference in normalized fluorescence output. We further characterize the expression- and burden profiles of each vector and demonstrate their use for the optimization of growth-coupled enzyme expression. The optimal expression of adhE (encoding alcohol dehydrogenase) for ethanol-dependent growth of Escherichia coli is determined using randomized Golden Gate Assembly, creating a diverse library of constructs with varying expression strengths and plasmid copy numbers. Through selective growth experiments, we show that relatively low expression levels of adhE facilitated optimal growth using ethanol as the sole carbon source, demonstrating the importance of adding low-copy vectors to the MoClo vector repertoire. This study emphasizes the importance of varying vector copy numbers in selection experiments to balance expression levels and burden, ensuring accurate identification of optimal conditions for growth. The vectors developed in this work are publicly available via Addgene (catalog #217582-217609).

Optimization of biohydrogen production by modifying metabolic pathways in Clostridium Thermocellum

The ever-escalating energy crisis of disruptions in the supply of fossil fuels suggests the need for alternative means with sustainable energy sources. Of these, biohydrogen stands out as promising because it is clean and efficient. Our research aims to provide strategies for fine-tuning the biohydrogen production process using the dark fermentation pathway of Clostridium. Thermocellum since it efficiently degrades cellulose under anaerobic conditions and has high-temperature tolerance, improving hydrogen yield. Sugarcane bagasse enforces this integration by providing abundant resources at little cost, which can support circular economies. However, such problems as ethanol toxicity to C. Thermocellum require genetic modifications to enhance production efficiency. The plasmid design overexpresses NADH-dependent [FeFe] hydrogenase, knocks out ethanol production pathways, and hence maximizes biohydrogen output. The design of the plasmid mainly uses Golden gate assembly and homologous recombination to achieve gene overexpression and deletion, and restriction sites were designed for virtual digestion in plasmid verification.

A point mutation in the pepper veinal mottle virus 6K1 protein yields a stable attenuated strain for engineering virus resistance in pepper plants

A total of 164 viruses have been identified in peppers worldwide. To combat viruses, pathogen-derived resistance (PDR) has been employed by expressing a viral genomic segment or a viral protein in host plants. Unfortunately, peppers are recalcitrant to genetic transformation and regeneration. An alternative strategy is to generate PDR in pepper plants by using an attenuated virus-based expression vector. In a previous study on the function of pepper veinal mottle virus (PVMV) 6K1 protein, we mutated a conserved Ala with Arg (A15R), and a second mutation R15G was observed in virus progeny. In this study, we demonstrated that mutating Ala to Gly (A15G) in the 6K1 yield a stable attenuated strain (named PVMV-G-A15G). PVMV-G-A15G can cross-protect pepper plants against a severe strain of PVMV and its close relative, chilli veinal mottle virus. We then engineered PVMV-G-A15G into a viral expression vector that has the Golden Gate cloning feature. To examine if the PVMV vector can be used to mediate PDR, we inserted the complete CP sequence of potato virus X (PVX) into the engineered PVMV-G-A15G. Pre-inoculated plants with the recombinant PVMV-G-A15G that carries the PVX CP sequence are resistant to PVX infection. Taken together, our research demonstrates that the attenuated PVMV can be employed for delivering target virus sequence to mediate PDR.

Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids Using Golden Gate Cloning.

Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen.

Golden Gate Cloning of MoClo Standard Parts.

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level -1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module.

Golden Gate Cloning in Actinobacteria: Opportunities and Challenges.

As we exploit biological machineries and circuits to redesign nature, it is just important to use efficient cloning strategies and methods to heterologously express the resulting DNA constructs. Golden Gate cloning allows the assembly of multiple fragments in a single reaction, making the process efficient and seamless. Although Golden Gate strategies have already been employed for different organisms, it is still not well-established for Actinobacteria. Here, we describe methods for Golden Gate cloning and how it can be utilized for Actinobacteria.

Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing.

Golden Gate cloning has revolutionized synthetic biology. Its concept of modular, highly characterized libraries of parts that can be combined into higher order assemblies allows engineering principles to be applied to biological systems. The basic parts, typically stored in Level 0 plasmids, are sequence validated by the method of choice and can be combined into higher order assemblies on demand. Higher order assemblies are typically transcriptional units, and multiple transcriptional units can be assembled into multi-gene constructs. Higher order Golden Gate assembly based on defined and validated parts usually does not introduce sequence changes. Therefore, simple validation of the assemblies, e.g., by colony polymerase chain reaction (PCR) or restriction digest pattern analysis is sufficient. However, in many experimental setups, researchers do not use defined parts, but rather part libraries, resulting in assemblies of high combinatorial complexity where sequencing again becomes mandatory. Here, we present a detailed protocol for the use of a highly multiplexed dual barcode amplicon sequencing using the Nanopore sequencing platform for in-house sequence validation. The workflow, called DuBA.flow, is a start-to-finish procedure that provides all necessary steps from a single colony to the final easy-to-interpret sequencing report.

MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering.

Photosynthetic microalgae are promising green cell factories for the sustainable production of high-value chemicals and biopharmaceuticals. The chloroplast organelle is being developed as a chassis for synthetic biology as it contains its own genome (the plastome) and some interesting advantages, such as high recombinant protein titers and a diverse and dynamic metabolism. However, chloroplast engineering is currently hampered by the lack of standardized cloning tools and Design-Build-Test-Learn workflows to ease genomic and metabolic engineering. The MoClo (Modular Cloning) toolkit based on Golden Gate assembly was recently developed in the model eukaryotic green microalgae Chlamydomonas reinhardtii to facilitate nuclear transformation and engineering. Here, we present MoCloro as an extension of the MoClo that allows chloroplast genome engineering. Briefly, a Golden Gate-compatible chloroplast transformation vector (pWF.K.4) was constructed, which contains homologous arms for integration at the petA site in the plastome. A collection of standardized parts (promoters, terminators, reporter and selection marker genes) was created following the MoClo syntax to enable easy combinatorial assembly of multi-cassettes in the destination pWF.K.4 vector. The functionality of the biobricks was assayed by constructing and assessing the expression of several multigenic constructs. Finally, a generic vector pK4 was constructed for easy Golden Gate cloning of 5' and 3' homologous arms, allowing targeting at alternative plastome integration sites. This work represents a significant advancement in technology aimed at facilitating more efficient and rapid chloroplast transformation and engineering of green microalgae.

Golden Gate Assembly of Transcriptional Unit Libraries into a Rearrangeable Gene Cluster.

Both regulatory sequences and genome organization contribute to the production of diverse transcript isoforms, which can influence how genes, or sets of genes, are expressed. An efficient, modular approach is needed to generate the combinatorial complexity required to empirically test many combinations of different regulatory sequences and different gene orders. Golden Gate assembly provides such a tool for seamless one-pot cleavage and ligation, by using type IIS restriction enzymes, which cleave outside of their recognition site. In addition to reducing the number of steps, this one-pot reaction can improve correct assemblies by the continued cleavage of self-ligation products that retain the recognition site. Switching the specific restriction enzyme used between steps allows for modular assembly of several units. A protocol to perform modular assemblies with two type IIS restriction enzymes, namely BsaI-v2-HF and BsmBI-v2, is described here. This protocol includes a description for generating destination vectors that add loxPsym sites between transcriptional units, allowing for diversification of gene order, orientation, and spacing.

BEVA2.0: modular assembly of golden gate-compatible vectors with expanded utility for genetic engineering.

This expansion for the modular vector assembly platform BEVA (Bacterial Expression Vector Archive) introduces 11 new BEVA parts including two new cloning site variants, two new antibiotic resistance modules, three new origins of replication, and four new accessary modules. As a result, the modular system is now doubled in size and expanded in its capacity to produce diverse replicating plasmids. Furthermore, it is now amenable to genetic engineering methods involving genome-manipulation of target strains through deletions or integrations. In addition to introducing the new modules, we provide several BEVA-derived Golden Gate cloning plasmids that are used to validate parts and that may be useful for genetic engineering of proteobacteria and other bacteria. We also introduce new parts to allow compatibility with the CIDAR MoClo parts libraries.

A systematic screening assay identifies efficient small guide RNAs for CRISPR activation.

CRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy. However, there is a lack of experimental validation tools for selecting efficient sgRNAs for downstream applications. Here, we present a screening assay capable of identifying efficient single- and double sgRNAs through fluorescence quantification in vitro. In addition, we provide a tailored Golden Gate cloning workflow for streamlined incorporation of selected sgRNA candidates into lentiviral (LVs) or adeno-associated viral vectors (AAVs). The overall workflow was validated using therapeutically relevant genes for neurodegenerative diseases, including Tfeb, Adam17, and Sirt1. The most efficient sgRNAs also demonstrated activation of endogenous gene expression at mRNA level. Correlation analysis of gene activation relative to sgRNA binding site distance to transcription start-site or nearby transcription factor binding sites failed to detect common characteristics influencing gene activation in the selected promoter regions. This data demonstrates the potential of the screening assay to identify functionally efficient sgRNA candidates across multiple genes along with streamlined cloning of viral vectors and may assist in accelerating future developments of CRISPRa-focused applications.

Design of a Golden Gate Cloning Plasmid for the Generation of a Chimeric Virus-Like Particle-Based Subunit Vaccine Against Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is a pervasive pathogen in the swine industry, leading to a spectrum of disorders known as porcine circovirus associated diseases (PCVAD). The PCV2 Cap protein contains critical antigenic epitopes and is the primary target for vaccine development. Current vaccines include inactivated viral particles and virus-like particles (VLPs), with experimental vaccines exploring various innovative approaches. This study introduces a novel Golden Gate assembly-based, Escherichia coli expression destination plasmid, pPRSVCP-E18, designed for the in-frame insertion of short peptide-coding DNA sequences into a highly antigenic site of Papaya ringspot virus (PRSV) coat protein (CP). We successfully cloned four PCV2 Cap peptides into this plasmid, expressed the chimeric CP proteins in E. coli under flask and bioreactor conditions, and assembled them into VLPs. These VLPs, when adjuvated and administered to BALB/c mice, elicited a specific IgG response against the PCV2 Cap peptides. Our findings demonstrate the plasmid's high efficiency for Golden Gate cloning and its potential in developing subunit vaccines against PCV2, contributing to the sustainable control of PCVAD in the swine industry.