Artificial Chromosome Research Articles

Microcell-mediated chromosome transfer between non-identical human iPSCs

Microcell-mediated chromosome transfer (MMCT) is anticipated as a unique strategy to manipulate numbers of chromosomes, including the generation of hyperaneuploidy syndrome models with human induced pluripotent stem cells (hiPSCs). Mouse A9/Chinese hamster ovary (CHO) cell libraries of human monochromosomal hybrids as chromosome donor cells frequently show chromosomal rearrangement in the components. The generation of a new A9/CHO library is time-consuming and laborious. Here, we developed an MMCT method using hiPSCs as chromosome donor and recipient cells, through micronucleation using paclitaxel and reversine. Membrane fusion during the MMCT was mediated through interactions between the ecotropic viral envelope transiently expressed in chromosome donor cells and mCAT-1 in chromosome recipient cells. This approach involved tagging Chr21 and ChrY by CRISPR/Cas9 and transferring human/mouse artificial chromosomes, Chr21, ChrX, and ChrY, wherein there are no previous reports demonstrating a full-length introduction. Thus, a strategy that combined CRISPR/Cas9-mediated chromosome tagging and MMCT from hiPSCs as chromosome donor cells to hiPSCs as recipient cells systematically produced isogenic disease model hiPSCs with hyperaneuploidy. This approach allows the study of rare diseases and promises to provide new insights into early developmental mechanisms by introducing a comprehensive set of influential chromosomes/regions into hiPSCs.

Enabling whole pathway reconstruction using artificial chromosomes.

Enabling whole pathway reconstruction using artificial chromosomes.

Artificial Chromosomes: Unlocking the Potential of Genetic Engineering

Artificial Chromosomes: Unlocking the Potential of Genetic Engineering

The Arabidopsis T-DNA mutant SALK_008491 carries a 14-kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress.

In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light‐dependent reaction and CO2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer‐DNA (T‐DNA) insertion mutant line SALK_008491, previously known as nhd1‐1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non‐photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2 pool, we identified an ~14‐kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1. Besides NHD1, which encodes for a putative plastid Na+/H+ antiporter, the stromal NAD‐dependent D‐3‐phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow‐up studies employing respective single mutants and complementation with overlapping transformation‐competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion‐carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T‐DNA lines. Although second‐site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T‐DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.

A new era of mass data storage in artificial chromosome.

A new era of mass data storage in artificial chromosome.

Historical DNA Manipulation Overview.

The history of DNA manipulation for the creation of genetically modified animals began in the 1970s, using viruses as the first DNA molecules microinjected into mouse embryos at different preimplantation stages. Subsequently, simple DNA plasmids were used to microinject into the pronuclei of fertilized mouse oocytes and that method became the reference for many years. The isolation of embryonic stem cells together with advances in genetics allowed the generation of gene-specific knockout mice, later on improved with conditional mutations. Cloning procedures expanded the gene inactivation to livestock and other non-model mammalian species. Lentiviruses, artificial chromosomes, and intracytoplasmic sperm injections expanded the toolbox for DNA manipulation. The last chapter of this short but intense history belongs to programmable nucleases, particularly CRISPR-Cas systems, triggering the development of genomic-editing techniques, the current revolution we are living in.

Huntington's Disease Gene Hunters: An Expanding Tale.

Huntington's Disease Gene Hunters: An Expanding Tale.

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.

Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability

BackgroundCentromeric regions of human chromosomes contain large numbers of tandemly repeated α-satellite sequences. These sequences are covered with constitutive heterochromatin which is enriched in trimethylation of histone H3 on lysine 9 (H3K9me3). Although well studied using artificial chromosomes and global perturbations, the contribution of this epigenetic mark to chromatin structure and genome stability remains poorly known in a more natural context.ResultsUsing transcriptional activator-like effectors (TALEs) fused to a histone lysine demethylase (KDM4B), we were able to reduce the level of H3K9me3 on the α-satellites repeats of human chromosome 7. We show that the removal of H3K9me3 affects chromatin structure by increasing the accessibility of DNA repeats to the TALE protein. Tethering TALE-demethylase to centromeric repeats impairs the recruitment of HP1α and proteins of Chromosomal Passenger Complex (CPC) on this specific centromere without affecting CENP-A loading. Finally, the epigenetic re-writing by the TALE-KDM4B affects specifically the stability of chromosome 7 upon mitosis, highlighting the importance of H3K9me3 in centromere integrity and chromosome stability, mediated by the recruitment of HP1α and the CPC.ConclusionOur cellular model allows to demonstrate the direct role of pericentromeric H3K9me3 epigenetic mark on centromere integrity and function in a natural context and opens interesting possibilities for further studies regarding the role of the H3K9me3 mark.

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.

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.

Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates.

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

CReasPy-Cloning: A Method for Simultaneous Cloning and Engineering of Megabase-Sized Genomes in Yeast Using the CRISPR-Cas9 System.

Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.

Histone H3K9 and H4 Acetylations and Transcription Facilitate the Initial CENP-AHCP\u22123 Deposition and De Novo Centromere Establishment in Caenorhabditis elegans Artificial Chromosomes

BackgroundThe 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.ResultsDNA 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.ConclusionsAcetylated 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.

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.

An Artificial Human Optimization Algorithm Titled Human Thinking Particle Swarm Optimization

Artificial Human Optimization is a latest field proposed in December 2016. Just like artificial Chromosomes are agents for Genetic Algorithms, similarly artificial Humans are agents for Artificial Human Optimization Algorithms. Particle Swarm Optimization is very popular algorithm for solving complex optimization problems in various domains. In this paper, Human Thinking Particle Swarm Optimization (HTPSO) is proposed by applying the concept of thinking of Humans into Particle Swarm Optimization. The proposed HTPSO algorithm is tested by applying it on various benchmark functions. Results obtained by HTPSO algorithm are compared with Particle Swarm Optimization algorithm.

Faithful Artificial Chromosome Propagation Using Spermatogonial Stem Cells

Artificial chromosomes are useful in making functional vectors for very large genes, studying chromosome physiology, and modeling chromosomal disorders. Shinohara et al. have succeeded for the first time in creating transchromosomic mice by manipulating spermatogonial stem cells (SSCs), which exhibited superior chromosomal stability compared with embryonic stem cells (ESCs).

Genetic evidence for functional interaction of Smc5/6 complex and Top1 with spatial frequency of replication origins required for maintenance of chromosome stability

Replication of linear chromosomes is facilitated by firing of multiple replication origins that ensures timely duplication of the entire chromosome. The Smc5/6 complex is thought to play an important role in replication by its involvement in the restart of collapsed replication forks. Here, we present genetic evidence for functional interaction between replication origin distribution and two subunits of the Smc5/6 complex, Smc6 and Mms21, as well as Top1. An artificial chromosome that has a long arm having low origin density (5ori∆YAC) is relatively unstable compared to the YAC having normal origin distribution in wild-type cells, but is partially stabilized in smc6-56 and top1∆ mutants. While a SUMO-ligase-deficient mutant of Mms21 does not affect stability of the 5ori∆YAC by itself, in combination with top1∆, the 5ori∆YAC is destabilized as evidenced by increased chromosome loss frequency in the mms21∆sl top1∆ double mutant. Likewise, the smc6-56 top1∆ double mutant also exhibits enhanced destabilization of the 5ori∆YAC compared to either single mutant. Such an increase in chromosome loss is not observed for a similar YAC that retains the original replication origins and normal origin distribution on the long arm, in either double mutant having the mms21∆sl or smc6-56 mutations in combination with top1∆. Our findings reveal a requirement for the Smc5/6 complex, including Mms21/Nse2 mediated sumoylation, and topoisomerase-1 (Top1), for maintaining stability of a chromosome having low origin density and suggest a functional cooperation between the Smc5/6 complex and Top1 in maintenance of topologically challenged chromosomes prone to replication fork collapse or accumulation of torsional stress.

Artificial Chromosomes and Strategies to Initiate Epigenetic Centromere Establishment.

In recent years, various synthetic approaches have been developed to address the question of what directs centromere establishment and maintenance. In this chapter, we will discuss how approaches aimed at constructing synthetic centromeres have co-evolved with and contributed to shape the theory describing the determinants of centromere identity. We will first review lessons learned from artificial chromosomes created from "naked" centromeric sequences to investigate the role of the underlying DNA for centromere formation. We will then discuss how several studies, which applied removal of endogenous centromeres or over-expression of the centromere-specific histone CENP-A, helped to investigate the contribution of chromatin context to centromere establishment. Finally, we will examine various biosynthetic approaches taking advantage of targeting specific proteins to ectopic sites in the genome to dissect the role of many centromere-associated proteins and chromatin modifiers for centromere inheritance and function. Together, these studies showed that chromatin context matters, particularly proximity to heterochromatin or repetitive DNA sequences. Moreover, despite the important contribution of centromeric DNA, the centromere-specific histone H3-variant CENP-A emerges as a key epigenetic mark to establish and maintain functional centromeres on artificial chromosomes or at ectopic sites of the genome.

Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi.

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.