Engineering better artificial chromosomes.

Constructing human artificial chromosomes in yeast avoids unintended multimerization.

Plant Artificial Chromosomes: Construction and Transformation.

With the decline of cultivated land and increase of the population in recent years, an agricultural revolution is urgently needed to produce more food to improve the living standards of humans. As one of the foundations of synthetic biology, artificial chromosomes hold great potential for advancing crop improvement. They offer opportunities to increase crop yield and quality, while enhancing crop resistance to disease. The progress made in plant artificial chromosome technology enables selective modification of existing chromosomes or the synthesis of new ones to improve crops and study gene function. However, current artificial chromosome technologies still face limitations, particularly in the synthesis of repeat sequences and the transformation of large DNA fragments. In this review, we will introduce the structure of plant centromeres, the construction of plant artificial chromosomes, and possible methods for transforming large fragments into plant cells.

Formation of artificial chromosomes in Caenorhabditis elegans and analyses of their segregation in mitosis, DNA sequence composition and holocentromere organization.

To investigate how exogenous DNA concatemerizes to form episomal artificial chromosomes (ACs), acquire equal segregation ability and maintain stable holocentromeres, we injected DNA sequences with different features, including sequences that are repetitive or complex, and sequences with different AT-contents, into the gonad of Caenorhabditis elegans to form ACs in embryos, and monitored AC mitotic segregation. We demonstrated that AT-poor sequences (26% AT-content) delayed the acquisition of segregation competency of newly formed ACs. We also co-injected fragmented Saccharomyces cerevisiae genomic DNA, differentially expressed fluorescent markers and ubiquitously expressed selectable marker to construct a less repetitive, more complex AC. We sequenced the whole genome of a strain which propagates this AC through multiple generations, and de novo assembled the AC sequences. We discovered CENP-AHCP-3 domains/peaks are distributed along the AC, as in endogenous chromosomes, suggesting a holocentric architecture. We found that CENP-AHCP-3 binds to the unexpressed marker genes and many fragmented yeast sequences, but is excluded in the yeast extremely high-AT-content centromeric and mitochondrial DNA (> 83% AT-content) on the AC. We identified A-rich motifs in CENP-AHCP-3 domains/peaks on the AC and on endogenous chromosomes, which have some similarity with each other and similarity to some non-germline transcription factor binding sites.

RbAp46/48LIN-53 and HAT-1 are required for initial CENP-AHCP-3 deposition and de novo holocentromere formation on artificial chromosomes in Caenorhabditis elegans embryos.

Foreign DNA microinjected into the Caenorhabditis elegans syncytial gonad forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Short, linear DNA fragments injected concatemerize into high molecular weight (HMW) DNA arrays that are visible as punctate DAPI-stained foci in oocytes, and they undergo chromatinization and centromerization in embryos. The inner centromere, inner kinetochore and spindle checkpoint components, including AIR-2, CENP-AHCP-3, Mis18BP1KNL-2 and BUB-1, respectively, assemble onto the nascent ACs during the first mitosis. The DNA replication efficiency of ACs improves over several cell cycles, which correlates with the improvement of kinetochore bi-orientation and proper segregation of ACs. Depletion of condensin II subunits, like CAPG-2 and SMC-4, but not the replicative helicase component, MCM-2, reduces de novo CENP-AHCP-3 level on nascent ACs. Furthermore, H3K9ac, H4K5ac and H4K12ac are highly enriched on newly chromatinized ACs. RbAp46/48LIN-53 and HAT-1, which affect the acetylation of histone H3 and H4, are essential for chromatinization, de novo centromere formation and segregation competency of nascent ACs. RbAp46/48LIN-53 or HAT-1 depletion causes the loss of both CENP-AHCP-3 and Mis18BP1KNL-2 initial deposition at de novo centromeres on ACs. This phenomenon is different from centromere maintenance on endogenous chromosomes, where Mis18BP1KNL-2 functions upstream of RbAp46/48LIN-53.

Characterization and use of Tetrahymena thermophila artificial chromosome 2 (TtAC2) constructed by biomimetic of macronuclear rDNA minichromosome.

Efficient expression vectors for unicellular ciliate eukaryotic Tetrahymena thermophila are still needed in recombinant biology and biotechnology applications. Previously, the construction of the T. thermophila Macronuclear Artificial Chromosome 1 (TtAC1) vector revealed additional needs for structural improvements such as better in vivo stability and maintenance as a recombinant protein expression platform. In this study, we designed an efficiently maintained artificial chromosome by biomimetic of the native macronuclear rDNA minichromosome. TtAC2 was constructed by sequential cloning of subtelomeric 3'NTS region (1.8 kb), an antibiotic resistance gene cassette (2 kb neo4), a gene expression cassette (2 kb TtsfGFP), rDNA coding regions plus a dominant C3 origin sequence (10.3 kb), and telomeres (2.4 kb) in a pUC19 backbone plasmid (2.6 kb). The 21 kb TtAC2 was characterized using fluorescence microscopy, qPCR, western blot and Southern blot after its transformation to vegetative T. thermophila CU428.2 strain, which has a recessive B origin allele. All experimental data show that circular or linear forms of novel TtAC2 were maintained as free replicons in T. thermophila macronucleus with or without antibiotic treatment. Notably, TtAC2 carrying strains expressed a TtsfGFP marker protein, demonstrating the efficacy and functionality of the protein expression platform. We show that TtAC2 is functionally maintained for more than two months, and can be efficiently used in recombinant DNA, and protein production applications.

Molecular organization of recombinant human-Arabidopsis chromosomes in hybrid cell lines.

Although plants and animals are evolutionarily distant, the structure and function of their chromosomes are largely conserved. This allowed the establishment of a human-Arabidopsis hybrid cell line in which a neo-chromosome was formed by insertion of segments of Arabidopsis chromosomes into human chromosome 15. We used this unique system to investigate how the introgressed part of a plant genome was maintained in human genetic background. The analysis of the neo-chromosome in 60- and 300-day-old cell cultures by next-generation sequencing and molecular cytogenetics suggested its origin by fusion of DNA fragments of different sizes from Arabidopsis chromosomes 2, 3, 4, and 5, which were randomly intermingled rather than joined end-to-end. The neo-chromosome harbored Arabidopsis centromeric repeats and terminal human telomeres. Arabidopsis centromere wasn't found to be functional. Most of the introgressed Arabidopsis DNA was eliminated during the culture, and the Arabidopsis genome in 300-day-old culture showed significant variation in copy number as compared with the copy number variation in the 60-day-old culture. Amplified Arabidopsis centromere DNA and satellite repeats were localized at particular loci and some fragments were inserted into various positions of human chromosome. Neo-chromosome reorganization and behavior in somatic cell hybrids between the plant and animal kingdoms are discussed.

A RAGE Based Strategy for the Genome Engineering of the Human Respiratory Pathogen Mycoplasma pneumoniae.

Genome engineering of microorganisms has become a standard in microbial biotechnologies. Several efficient tools are available for the genetic manipulation of model bacteria such as Escherichia coli and Bacillus subtilis, or the yeast Saccharomyces cerevisiae. Difficulties arise when transferring these tools to nonmodel organisms. Synthetic biology strategies relying on genome transplantation (GT) aim at using yeast cells for engineering bacterial genomes cloned as artificial chromosomes. However, these strategies remain unsuccessful for many bacteria, including Mycoplasma pneumoniae (MPN), a human pathogen infecting the respiratory tract that has been extensively studied as a model for systems biology of simple unicellular organisms. Here, we have designed a novel strategy for genome engineering based on the recombinase-assisted genomic engineering (RAGE) technology for editing the MPN genome. Using this strategy, we have introduced a 15 kbp fragment at a specific locus of the MPN genome and replaced 38 kbp from its genome by engineered versions modified either in yeast or in E. coli. A strain harboring a synthetic version of this fragment cleared of 13 nonessential genes could also be built and propagated in vitro. These strains were depleted of known virulence factors aiming at creating an avirulent chassis for SynBio applications. Such a chassis and technology are a step forward to build vaccines or deliver therapeutic compounds in the lungs to prevent or cure respiratory diseases in humans.

Heterologous Expression of the Unusual Terreazepine Biosynthetic Gene Cluster Reveals a Promising Approach for Identifying New Chemical Scaffolds.

Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates that FAC-based approaches targeting unusual biosynthetic machinery provide a promising avenue forward for targeted discovery of novel scaffolds and their biosynthetic enzymes, and it also represents another example of a biosynthetic gene cluster "repurposing" a primary metabolic enzyme to diversify its secondary metabolite arsenal.IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the numerous strategies fungi employ for diversifying and protecting themselves against secondary metabolites. The observations in this study set the stage for further in-depth studies into the function of duplicated IDOs present in fungal biosynthetic gene clusters and presents a strategy for accessing the biosynthetic potential of gene clusters containing duplicated primary metabolic genes.

Charting the path to fully synthetic plant chromosomes.

The concepts of synthetic biology have the potential to transform plant genetics, both in how we analyze genetic pathways and how we transfer that knowledge into useful applications. While synthetic biology can be applied at the level of the single gene or small groups of genes, this commentary focuses on the ultimate challenge of designing fully synthetic plant chromosomes. Engineering at this scale will allow us to manipulate whole genome architecture and to modify multiple pathways and traits simultaneously. Advances in genome synthesis make it likely that the initial phases of plant chromosome construction will occur in bacteria and yeast. Here I discuss the next steps, including specific ways of overcoming technical barriers associated with plant transformation, functional centromere design, and ensuring accurate meiotic transmission.

Gene expressing human artificial chromosome vectors: Advantages and challenges for gene therapy.

After the construction of genomic libraries with yeast artificial chromosomes in the late 1980's for gene isolation and expression studies in cells, human artificial chromosomes were then a natural development in the 1990's, based on the same principles of formation requiring centromeric sequences for generating functional artificial chromosomes. Over the past twenty years, they became a useful research tool for understanding human chromosome structure and organization, and important vectors for expression of large genes and gene loci and the regulatory regions for full expression. Now they are being modified and developed for gene therapy both ex vivo and in vivo. The advantages of using HAC vectors are that they remain autonomous and behave as a normal chromosome. They are attractive for therapy studies without the harmful consequences of integration of exogenous DNA into host chromosomes. HAC vectors are also the only autonomous stable vectors that accommodate large sequences (>100 kb) compared to other vectors. The challenges of manipulating these vectors for efficient delivery of genes into human cells is still ongoing, but we have made advances in transfer of gene expressing HAC vectors using the helper free (HF) amplicon vector technology for generating de novo HAC in human cells. Efficient multigene delivery was successfully achieved following simultaneous infection with two HF amplicons in a single treatment and the input DNA recombined to form a de novo HAC. Potentially several amplicons containing gene expressing HAC vectors could be transduced simultaneously which would increase the gene loading capacity of the vectors for delivery and studying full expression in human cells.

A novel Xist RNA-mediated chromosome inactivation model using a mouse artificial chromosome.

OBJECTIVE: To develop a mouse artificial chromosome (MAC) carrying the mouse Xist gene (X-inactive specific transcript; Xist-MAC) as a systematic in vitro approach for investigating Xist RNA-mediated chromosome inactivation. RESULTS: Ectopic expression of the Xist gene in CHO cells led to the accumulation of Xist RNA in cis on the MAC. In addition, the introduction of Xist-MAC to embryonic stem cells from male mice via microcell-mediated chromosome transfer resulted in the accumulation of Xist RNA in cis on the MAC. Chromosomal inactivation was observed in the differentiated state. Moreover, this phenomenon was accompanied by the epigenetic modification of H3K27 trimethylation. CONCLUSIONS: We successfully generated a novel chromosome inactivation model, Xist-MAC, which will provide a valuable tool for the screening and functional analysis of X chromosome inactivation-related genes and proteins.

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform.

Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells.

A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a breakage-fusion-bridge cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology.

An assessment of transgenomics as a tool for gene discovery in Populus euphratica Oliv.

KEY MESSAGE: Transgenomics for gene discovery in Populus euphratica. Transgenomics, a member of the omics family of methodologies, is characterized as the introduction of DNA from one organism into another on a genome-wide scale followed by the identification of recipients with altered phenotypes. This strategy allows investigators to identify the gene(s) involved in these phenotypic changes. It is particularly promising for woody plants that have a long life cycle and for which molecular tools are limited. In this study, we constructed a large-insert binary bacterial artificial chromosome library of Populus euphratica, a stress-tolerant poplar species, which included 55,296 clones with average insert sizes of about 127 kb. To date, 1077 of the clones have been transformed into Arabidopsis thaliana via Agrobacterium by the floral dip method. Of these, 69 transgenic lines showed phenotypic changes represented by diverse aspects of plant form and development, 22 of which were reproducibly associated with the same phenotypic change. One of the clones conferring transgenic plants with increased salt tolerance, 002A1F06, was further analyzed and the 127,284 bp insert in this clone harbored eight genes that have been previously reported to be involved in stress resistance. This study demonstrates that transgenomics is useful in the study of functional genomics of woody plants and in the identification of novel gene(s) responsible for economically important traits. Thus, transgenomics can also be used for validation of quantitative trait loci mapped by molecular markers.

Large inserts for big data: artificial chromosomes in the genomic era.

The exponential increase in available microbial genome sequences coupled with predictive bioinformatic tools is underscoring the genetic capacity of bacteria to produce an unexpected large number of specialized bioactive compounds. Since most of the biosynthetic gene clusters (BGCs) present in microbial genomes are cryptic, i.e. not expressed under laboratory conditions, a variety of cloning systems and vectors have been devised to harbor DNA fragments large enough to carry entire BGCs and to allow their transfer in suitable heterologous hosts. This minireview provides an overview of the vectors and approaches that have been developed for cloning large BGCs, and successful examples of heterologous expression.

Histone H3K9 and H4 Acetylations and Transcription Facilitate the Initial CENP-AHCP-3 Deposition and De Novo Centromere Establishment in Caenorhabditis elegans Artificial Chromosomes.

BACKGROUND: The centromere is the specialized chromatin region that directs chromosome segregation. The kinetochore assembles on the centromere, attaching chromosomes to microtubules in mitosis. The centromere position is usually maintained through cell cycles and generations. However, new centromeres, known as neocentromeres, can occasionally form on ectopic regions when the original centromere is inactivated or lost due to chromosomal rearrangements. Centromere repositioning can occur during evolution. Moreover, de novo centromeres can form on exogenously transformed DNA in human cells at a low frequency, which then segregates faithfully as human artificial chromosomes (HACs). How centromeres are maintained, inactivated and activated is unclear. A conserved histone H3 variant, CENP-A, epigenetically marks functional centromeres, interspersing with H3. Several histone modifications enriched at centromeres are required for centromere function, but their role in new centromere formation is less clear. Studying the mechanism of new centromere formation has been challenging because these events are difficult to detect immediately, requiring weeks for HAC selection. RESULTS: DNA injected into the Caenorhabditis elegans gonad can concatemerize to form artificial chromosomes (ACs) in embryos, which first undergo passive inheritance, but soon autonomously segregate within a few cell cycles, more rapidly and frequently than HACs. Using this in vivo model, we injected LacO repeats DNA, visualized ACs by expressing GFP::LacI, and monitored equal AC segregation in real time, which represents functional centromere formation. Histone H3K9 and H4 acetylations are enriched on new ACs when compared to endogenous chromosomes. By fusing histone deacetylase HDA-1 to GFP::LacI, we tethered HDA-1 to ACs specifically, reducing AC histone acetylations, reducing AC equal segregation frequency, and reducing initial kinetochroe protein CENP-AHCP-3 and NDC-80 deposition, indicating that histone acetylations facilitate efficient centromere establishment. Similarly, inhibition of RNA polymerase II-mediated transcription also delays initial CENP-AHCP-3 loading. CONCLUSIONS: Acetylated histones on chromatin and transcription can create an open chromatin environment, enhancing nucleosome disassembly and assembly, and potentially contribute to centromere establishment. Alternatively, acetylation of soluble H4 may stimulate the initial deposition of CENP-AHCP-3-H4 nucleosomes. Our findings shed light on the mechanism of de novo centromere activation.

Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

Development of a multiple-gene-loading method by combining multi-integration system-equipped mouse artificial chromosome vector and CRISPR-Cas9.

Mouse artificial chromosome (MAC) vectors have several advantages as gene delivery vectors, such as stable and independent maintenance in host cells without integration, transferability from donor cells to recipient cells via microcell-mediated chromosome transfer (MMCT), and the potential for loading a megabase-sized DNA fragment. Previously, a MAC containing a multi-integrase platform (MI-MAC) was developed to facilitate the transfer of multiple genes into desired cells. Although the MI system can theoretically hold five gene-loading vectors (GLVs), there are a limited number of drugs available for the selection of multiple-GLV integration. To overcome this issue, we attempted to knock out and reuse drug resistance genes (DRGs) using the CRISPR-Cas9 system. In this study, we developed new methods for multiple-GLV integration. As a proof of concept, we introduced five GLVs in the MI-MAC by these methods, in which each GLV contained a gene encoding a fluorescent or luminescent protein (EGFP, mCherry, BFP, Eluc, and Cluc). Genes of interest (GOI) on the MI-MAC were expressed stably and functionally without silencing in the host cells. Furthermore, the MI-MAC carrying five GLVs was transferred to other cells by MMCT, and the resultant recipient cells exhibited all five fluorescence/luminescence signals. Thus, the MI-MAC was successfully used as a multiple-GLV integration vector using the CRISPR-Cas9 system. The MI-MAC employing these methods may resolve bottlenecks in developing multiple-gene humanized models, multiple-gene monitoring models, disease models, reprogramming, and inducible gene expression systems.

Human and mouse artificial chromosome technologies for studies of pharmacokinetics and toxicokinetics.

In the earliest stage of drug discovery/development, various cell-based models and animal models were used for the prediction of human pharmacokinetics and toxicokinetics. Unfortunately, drugs under development are often discontinued because their nonclinical results do not extrapolate to human clinical studies in relation to either safety or efficacy. Therefore, it is important to improve the time- and cost-effectiveness of drug development. This might be achieved by developing new technologies including pharmacokinetics and toxicokinetics models that use human and mouse artificial chromosome vectors (HACs/MACs). HACs/MACs are unique vectors with several advantages: 1) independent maintenance, 2) defined copy number and mitotically stable, 3) no silencing of the transgene, and 4) no limitation of DNA insertion size. This review provides information on the advantages and examples of the utility of various models based on the recent advances in HAC/MAC technologies, including multifunctional cell-based models for assaying drug-drug interactions, bidirectional permeability, and cytotoxicity, as well as fully genetically humanized mouse models. We also discuss the future prospects of these technologies to advance drug discovery. In summary, these technologies offer advantages over current conventional models and should improve the success rate of drug development related to efficacy and safety for humans.