Large DNA Constructs Research Articles

A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster.

The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct capture of genome DNAs, by combining CRISPR and Gibson assembly. We demonstrate this method with the ability of cloning large DNA fragments ranging from 30 to 77 kb from various host genomes, achieving a near 100% cloning fidelity for DNA fragments below 50 kb. We next demonstrate this method by the cloning of a 40 kb fragment from Streptomyces ceruleus A3(2), which is rich in BGCs for natural products; and used this method cloning the 40 kb fengycin synthetic gene cluster from B. subtilis 168, encoding for a class of peptides with bioactivity. This method provides efficient and simple opportunities for assembling large DNA constructs from distant sources.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

CREEPY: CRISPR-mediated editing of synthetic episomes in yeast.

Use of synthetic genomics to design and build 'big' DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. Saccharomyces cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, introducing designer variations to episomal assemblies with high efficiency and fidelity remains challenging. Here we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, a method for rapid engineering of large synthetic episomal DNA constructs. We demonstrate that CRISPR editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes. We optimize CREEPY for efficient and precise multiplex editing of >100 kb yeast episomes, providing an expanded toolkit for synthetic genomics.

Mechanical deformation behaviors and structural properties of ligated DNA crystals

DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied extensively, the deformation behaviors and structural properties of self-assembled architectures are not well understood. This is partly due to the small dimensions and limited experimental methods available. DNA crystals are macroscopic crystalline structures assembled from nanoscale motifs via sticky-end association. The large DNA constructs may thus be an ideal platform to study structural mechanics. Here, we investigate the fundamental mechanical properties and behaviors of ligated DNA crystals made of tensegrity triangular motifs. We perform coarse-grained molecular dynamics simulations and confirm the results with nanoindentation experiments using atomic force microscopy. We observe various deformation modes, including untension, linear elasticity, duplex dissociation, and single-stranded component stretch. We find that the mechanical properties of a DNA architecture are correlated with those of its components. However, the structure shows complex behaviors which may not be predicted by components alone and the architectural design must be considered.

Programmable RNA-Guided Large DNA Transgenesis by CRISPR/Cas9 and Site-Specific Integrase Bxb1.

The inability to insert large DNA constructs into the genome efficiently and precisely is a key challenge in genomic engineering. Random transgenesis, which is widely used, lacks precision, and comes with a slew of drawbacks. Lentiviral and adeno-associated viral methods are plagued by, respectively, DNA toxicity and a payload capacity of less than 5 kb. Homology-directed repair (HDR) techniques based on CRISPR-Cas9 can be effective, but only in the 1–5 kb range. In addition, long homology arms—DNA sequences that permit construct insertion—of lengths ranging from 0.5 to 5 kb are required by currently known HDR-based techniques. A potential new method that uses Cas9-guided transposases to insert DNA structures up to 10 kb in length works well in bacteria, but only in bacteria. Surmounting these roadblocks, a new toolkit has recently been developed that combines RNA-guided Cas9 and the site-specific integrase Bxb1 to integrate DNA constructs ranging in length from 5 to 43 kb into mouse zygotes with germline transmission and into human cells. This ground-breaking toolkit will give researchers a valuable resource for developing novel, urgently needed mouse and human induced pluripotent stem cell (hiPSC) models of cancer and other genetic diseases, as well as therapeutic gene integration and biopharmaceutical applications, such as the development of stable cell lines to produce therapeutic protein products.

Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design.

Large DNA constructs (>10 kb) are invaluable tools for genetic engineering and the development of therapeutics. However, the manufacture of these constructs is laborious, often involving multiple hierarchical rounds of preparation. To address this problem, we sought to test whether Golden Gate assembly (GGA), an in vitro DNA assembly methodology, can be utilized to construct a large DNA target from many tractable pieces in a single reaction. While GGA is routinely used to generate constructs from 5 to 10 DNA parts in one step, we found that optimization permitted the assembly of >50 DNA fragments in a single round. We applied these insights to genome construction, successfully assembling the 40 kb T7 bacteriophage genome from up to 52 parts and recovering infectious phage particles after cellular transformation. The assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene “landing pad” composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies > 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.

Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination.

Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a PHIS3–HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.

PhiC31 integrase for recombination-mediated single-copy insertion and genome manipulation in Caenorhabditis elegans.

Caenorhabditis elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination-mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual-color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

Stabilization of Poly (β-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon.

The administration of gene-editing tools has been proposed as a promising therapeutic approach for correcting mutations that cause diseases. Gene-editing tools, composed of relatively large plasmid DNA constructs that often need to be co-delivered with a guiding protein, are unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery, they are restricted by the size of the plasmid to carry. In this work, we describe a strategy for the stable encapsulation of the gene-editing tool piggyBac transposon into Poly (β-amino ester) nanoparticles (NPs). We propose a non-covalent and a covalent strategy for stabilization of the nanoformulation to slow down release kinetics and enhance intracellular delivery. We found that the formulation prepared by covalently crosslinking Poly (β-amino ester) NPs are capable to translocate into the cytoplasm and nuclei of human glioblastoma (U87MG) cells within 1 h of co-culturing, without the need of a targeting moiety. Once internalized, the nanoformulation dissociates, delivering the plasmid presumably as a response to the intracellular acidic pH. Transfection efficiency is confirmed by green fluorescence protein (GFP) expression in U87MG cells. Covalently stabilized Poly (β-amino ester) NPs are able to transfect ~55% of cells causing non-cytotoxic effects. The strategy described in this work may serve for the efficient non-viral delivery of other gene-editing tools.

Efficient Transgenesis in Caenorhabditis elegans Using Flp Recombinase-Mediated Cassette Exchange.

The application of CRISPR technology has greatly facilitated the creation of transgenic Caenorhabditis elegans lines. However, methods to insert multi-kilobase DNA constructs remain laborious even with these advances. Here, I describe a new approach for introducing large DNA constructs into the C. elegans genome at specific sites using a combination of Flp and Cre recombinases. The system utilizes specialized integrated landing sites that express GFP ubiquitously flanked by single loxP, FRT, and FRT3 sites. DNA sequences of interest are inserted into an integration vector that contains a sqt-1 self-excising cassette and FRT and FRT3 sites. Plasmid DNA is injected into the germline of landing site animals. Transgenic animals are identified as Rol progeny, and the sqt-1 marker is subsequently excised with heat shock Cre expression. Integration events were obtained at a rate of approximately one integration per three injected F0 animals-a rate substantially higher than any current approach. To demonstrate the robustness of the approach, I compared the efficiency of the Gal4/UAS, QF (and QF2)/QUAS, tetR(and rtetR)/tetO, and LexA/lexO bipartite expression systems by assessing expression levels in combinations of driver and reporter GFP constructs and a direct promoter GFP fusion each integrated at multiple sites in the genome. My data demonstrate that all four bipartite systems are functional in C. elegans Although the new integration system has several limitations, it greatly reduces the effort required to create single-copy insertions at defined sites in the C. elegans genome.

Universal loop assembly: open, efficient and cross-kingdom DNA fabrication.

Standardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a ‘common syntax’ that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in host-specific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe ‘universal Loop’ (uLoop) assembly, a system based on Loop assembly for use in potentially any organism of choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms, yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.

The AssemblX Toolkit for Reliable and User-Friendly Multigene Assemblies.

The implementation of complex cloning projects covering the assembly of entire biological pathways or large genetic circuits poses a major challenge in the field of biotechnology and synthetic biology, as such projects can be costly and time-consuming. To overcome these difficulties, we developed the software-assisted AssemblX toolkit, which allows even unexperienced users to design, build, and subsequently test large DNA constructs. Currently, AssemblX allows the assembly of up to 25 functional units (e.g., genes), from 75 or more subunits (e.g., promoters, coding sequences, terminators). At the first assembly level, AssemblX uses overlap-based, scar-free, and sequence-independent cloning methods. This allows the unrestricted design at the gene level without the need for laborious parts domestication. The standardized, polymerase chain reaction-free, and virtually sequence-independent assembly into multigene modules relies on rare cutting homing endonucleases and computationally optimized overlap sequences. Selection and marker switching strategies ensure an effective process, and the assembly product can be transferred to any desired expression host.

Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae.

A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5‐isomerase gene from Comamonas testosteronii, (c) a mutated steroid‐5β‐reductase gene from Arabidopsis thaliana, and (d) a steroid 21‐hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed “CARD II yeast”, was capable of producing 5β‐pregnane‐3β,21‐diol‐20‐one, a central intermediate in 5β‐cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast.

Targeted insertion of large DNA sequences by homology-directed repair or non-homologous end joining in engineered tobacco BY-2 cells using designed zinc finger nucleases.

Targeted integration of recombinant DNA fragments into plant genomes by DNA double‐strand break (DSB) repair mechanisms has become a powerful tool for precision engineering of crops. However, many targeting platforms require the screening of many transgenic events to identify a low number of targeted events among many more random insertion events. We developed an engineered transgene integration platform (ETIP) that uses incomplete marker genes at the insertion site to enable rapid phenotypic screening and recovery of targeted events upon functional reconstitution of the marker genes. The two marker genes, encoding neomycin phosphotransferase II (nptII) and Discosoma sp. red fluorescent protein (DsRed) enable event selection on kanamycin‐containing selective medium and subsequent screening for red fluorescent clones. The ETIP design allows targeted integration of donor DNA molecules either by homology‐directed repair (HDR) or non‐homologous end joining (NHEJ)‐mediated mechanisms. Targeted donor DNA integration is facilitated by zinc finger nucleases (ZFN). The ETIP cassette was introduced into Nicotiana tabacum BY‐2 suspension cells to generate target cell lines containing a single copy locus of the transgene construct. The utility of the ETIP platform has been demonstrated by targeting DNA constructs containing up to 25‐kb payload. The success rate for clean targeted DNA integration was up to 21% for HDR and up to 41% for NHEJ based on the total number of calli analyzed by next‐generation sequencing (NGS). The rapid generation of targeted events with large DNA constructs expands the utility of the nuclease‐mediated gene addition platform both for academia and the commercial sector.

MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology.

Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5' untranslated regions [5'UTRs], nine promoter:5'UTR fusions, 10 3'UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.

Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

A synthetic mitochondrial-based vector for therapeutic purposes

Delivery of large DNA constructs is necessary for combinatorial, anti-aging gene therapy. However, this is hindered by the lack of a non-inflammatory vector with sufficient packaging space and the ability to effectively spread through tissue. It is possible that a mitochondrion, altered to be able to secrete therapeutic proteins, could serve as an ideal gene delivery vector in this regard (for post-mitotic cells at least). Initial transfection of mitochondria with a therapeutic gene vector could be undertaken by MITO-Porter, i.e., a dual-layered fusogenic liposomal-based delivery system. Then, once a protein export pathway from the mitochondrial matrix to the cytoplasm is achieved via Sec translocon installation in the inner membrane (IM), three other components would be necessary for vector maintenance. First of all, fusion with other mitochondria must be prevented through mitofusin-cleaving protease expression on the outer membrane (OM). Second of all, mitophagy must be evaded via the expression of deubiquitylases (DUBs) on the OM. Third of all, free radical damage to the membrane over time could be prevented by shutting down electron transport chain (ETC) activity via destruction of the endogenous mitochondrial DNA (mtDNA) inside the vector – and expression of an ATP import channel to harvest sufficient energy from the host cell. The most crucial part of this plan would be to achieve Sec translocon installation in the IM. The hypothesis of this article is that Sec translocon installation into the IM may be achieved using one of a few molecular methods described herein. It is possible that it could simply occur through gene expression from a mitochondrial matrix-localized gene construct and spontaneous (or Oxa1-assisted) insertion into the mitochondrial IM. Alternatively, it could be incorporated into the MITO-Porter IM prior to fusion with patient-derived mitochondria. Lastly, it may be sent to the mitochondrial IM via the TIM/TOM complex, although hydrophobic membrane proteins may fold prematurely in the cytoplasm before reaching the mitochondrial TIM/TOM complex. Regardless, SecYEG, when present in the IM to a small degree initially, should suffice to install more copies of itself from a mitochondrial matrix-localized gene vector. After proteins (with a C-terminal tag and autotransporter domain) have been sent to the intermembrane space from the matrix in unfolded form, the endogenous SAM complex would suffice to export them to the OM, where the relevant (passenger) domain may be cleaved off by an orthogonal protease and released into the cytoplasm.

Single Day Construction of Multigene Circuits with 3G Assembly.

The ability to rapidly design, build, and test prototypes is of key importance to every engineering discipline. DNA assembly often serves as a rate limiting step of the prototyping cycle for synthetic biology. Recently developed DNA assembly methods such as isothermal assembly and type IIS restriction enzyme systems take different approaches to accelerate DNA construction. We introduce a hybrid method, Golden Gate-Gibson (3G), that takes advantage of modular part libraries introduced by type IIS restriction enzyme systems and isothermal assembly's ability to build large DNA constructs in single pot reactions. Our method is highly efficient and rapid, facilitating construction of entire multigene circuits in a single day. Additionally, 3G allows generation of variant libraries enabling efficient screening of different possible circuit constructions. We characterize the efficiency and accuracy of 3G assembly for various construct sizes, and demonstrate 3G by characterizing variants of an inducible cell-lysis circuit.

Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis

SummaryImproved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.