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Engineered minichromosomes in plants
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Engineered minichromosomes in plants James A. Birchler∗, Nathan C. Swyers Division of Biological Sciences, University of Missouri, 311 Tucker Hall, Columbia, MO, 65211-7400, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Artificial chromosomes Synthetic chromosomes Maize Telomere-mediated truncation Gene stacking Site specific recombinases
Artificial chromosome platforms are described in plants. Because the function of centromeres is largely epigenetic, attempts to produce artificial chromosomes with plant centromere DNA have failed. The removal of the centromeric sequences from the cell strips off the centromeric histone that is the apparent biochemical marker of centromere activity. Thus, engineered minichromosomes have been produced by telomere mediated chromosomal truncation. The introduction of telomere repeats will cleave the chromosome at the site of insertion and attach the accompanying transgenes in the process. Such truncation events have been documented in maize, Arabidopsis, barley, rice, Brassica and wheat. Truncation of the nonvital supernumerary B chromosome of maize is a favorite target but engineered minichromosomes derived from the normal A chromosomes have also been recovered. Transmission through mitosis of small chromosomes is apparently normal but there is loss during meiosis. Potential solutions to address this issue are discussed. With procedures now well established to produce the foundation for artificial chromosomes in plants, current efforts are directed at building them up to specification using gene stacking methods and editing techniques.
1. Introduction In the past several decades the amount of arable land on the planet has been decreasing while the human population has been increasing. If the human population wishes to improve its standard of living, or indeed to simply maintain it, agriculture will need a revolution in order to produce more food in those coming decades than has ever been produced before [1]. Agriculture has had a profound impact on the biosphere with a predicted loss of natural animal species consisting of one out of 8 of them in the coming decades [2]. Ideas to adapt to these changes include the use of synthetic biology utilizing the directed evolution of enzymatic functions and synthetic transcription factors to produce plants with new combinations of properties. Various efforts are presently in place to attempt to confer nitrogen fixation to the major cereal crops that provide the vast majority of calories to the world but at the expense of considerable fertilizer application. Other possibilities are programs to improve the efficiency of photosynthesis, to confer the efficient C4 photosynthesis to species with C3 such as rice, to sequester carbon dioxide in plants, to enhance the nutritional components of crops, to modify crops to withstand environmental challenges and to provide plants with resistances to insect, viral, bacterial and fungal pests. Through millennia, humans have modified plants via unwitting and intentional selection to bring civilization to its current state. Such activity must continue.
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Synthetic approaches coupled with natural variation, which must also be secured, promise to be an effective avenue for crop improvement. One contribution to such an effort would be the development of means to construct independent chromosomes, which might harbor a collection of plant modifications. Artificial chromosomes were first assembled in yeast [3] and was the paradigm for applications in other species. They were generated by the assembly of centromeres, telomeres, an origin of replication and a selectable marker. When reintroduced into yeast cells, they functioned autonomously. However, using that paradigm for plants has not been successful to date despite numerous attempts in various species. The transformation of DNA from known centromere regions of chromosomes only produces stable integrations in the most rigorously controlled experiments [4]. The apparent reason is that the function of centromeres in plants is basically epigenetic with the underlying DNA not being responsible for their activity [5]. Instead, centromere function appears to rely on the presence of the centromere specific variant of histone H3, whose location on the chromosome is perpetuated from one cell cycle to the next. The role of engineered minichromosomes/artificial chromosomes/ synthetic chromosomes in plants has garnered attention in the past few years for a variety of reasons [6–8]. Not only do they have potential as a facile experimental tool but they hold promise for a variety of practical applications. Current methodologies for plant transformation rely on random integrations into the genome that result in variable expression
Corresponding author. E-mail address: [email protected] (J.A. Birchler).
https://doi.org/10.1016/j.yexcr.2020.111852 Received 3 November 2019; Accepted 14 January 2020 0014-4827/ © 2020 Elsevier Inc. All rights reserved.
Please cite this article as: James A. Birchler and Nathan C. Swyers, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2020.111852
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in basically all genetic models and crop species. Indeed, in addition to maize, telomere mediated truncation has been reported in Arabidopsis [23,24], barley [25], rice [26], wheat [27], and Brassica [28]. When normal chromosomes are truncated an aneuploid condition is generated that might be detrimental to plant growth and vigor and any deletions of vital genes will abort the haploid gametophyte generation. Despite these considerations, various approaches can be used to produce minichromosomes and recover them. In maize, there is a supernumerary B chromosome that is nonvital [29]; thus, it is a convenient target for truncation [21]. A strategy is outlined in Fig. 1 for utilizing telomere-mediated chromosomal truncation to target a B chromosome in maize to create an engineered minichromosomes. Another approaches that have been used is to target a tetraploid as the starting material [26] that would diminish any aneuploid effect and cover any deficiencies in the gametophyte generation, which in this case would be diploid. An additional approach was to use a telotrisomic genotype of rice that contains an extra chromosome arm as the trisomic addition. Truncation of this chromosome would actually remove aneuploid effects and produced an engineered minichromosome in the process. Wheat is a natural allohexaploid that can tolerate nullisomic conditions so truncation of chromosomes can be recovered [27]. Also, the rye B chromosome has been transferred to wheat and could serve as a target. Despite these aids in recovering truncated chromosomes, it is possible to obtain them from diploid targets directly. In addition to adding telomere repeats to selectable marker constructs for transformation, one can simply perform a cobombardment of the marker cassette with free telomere repeats [30]. An example was recovered that removed both arms of a chromosome but included the addition of the marker cassette. Presumably, the removal of both arms occurred to create a functional monosomic condition but the doubling of the remaining normal homologue occurred during the tissue culture selection. This example illustrates that even using a diploid target it is possible to recover engineered minichromosomes. The recovery of small chromosomes to date has relied on the random integration of the truncating constructs. This approach has been sufficiently frequent to recover very small chromosomes with added genes. However, in the current era of extensive genome sequence data and the ability to edit specific sequences, it is conceivable that targeted truncation might be possible in the future. One can imagine that it could be possible to remove all of the endogenous genes from a chromosome leaving the centromere and added DNA to build the chromosome to specification. Such an approach would leave a natural centromere that has never left a plant cell to maintain its function but would not leave any genes in the minichromosome foundation.
states of the transgenes and are potentially mutagenic if they impact endogenous gene expression. An independent chromosome would avoid these effects as well as any complications of linkage to undesired endogenous genes when incorporating transgenes from one variety to another. Also, combining multiple unlinked transgenes for stacking traits quickly becomes quite complicated as the assembly of unlinked insertions builds. Autonomously built chromosomes from scratch would overcome these issues. 1.1. Epigenetic nature of centromeres Before proceeding to a description of producing engineered minichromosome in plants, a discussion of centromere functions is needed. The realization of the frequency with which centromeres in plants could become inactive came from experiments that generated functionally dicentric chromosomes repeatedly over the life cycle of maize [9]. Among the progeny of plants with this configuration were sequence dicentrics with only a single centromere with retention of the centromere specific centromeric H3 (CENH3). Once this realization became evident, many additional examples of inactive centromeres were realized [10,11]. Directed inactivation was achieved by tying together, via crossing over with a unique foldback chromosome, a normal large and a version of the same centromere that had been experimentally reduced in size [10,12]. Recombination to produce a tug of war resulted in the smaller centromere becoming inactive with loss of CENH3. The collective results of many examples of inactive centromeres indicates the inactivation can occur over a maximum of one or a few cell cycles and is perpetuated in the inactive state in perpetuity. The flip side observation indicating an epigenetic feature of plant centromeres is the repeated finding of de novo formed centromeres on chromatin fragments that are missing canonical centromeric DNA [13–15]. ChIP-Seq experiments using antibodies against the respective CENH3 performed on fragments with known origins confirmed that new centromere formation had occurred over unique sequences present in chromosome arms from which the fragment originated [14,15]. Numerous cases of de novo centromeres have now been determined and, in some cases, the timeframe of formation of the fragments had been documented, which demonstrated in these cases as well that they emerged over only one or a few cell cycles and then perpetuated over decades. Variation in the exact position of CENH3 in centromeric regions in different varieties of maize indicates that centromere inactivation and de novo formation is a natural occurrence on a regular basis [16]. 1.2. Telomere mediated chromosomal truncation
1.3. Behavior of small chromosomes in meiosis With the realization of the epigenetic nature of centromeres in plants, it became evident that the removal of centromeric DNA from cells and its reintroduction would be ineffective as a component of an artificial chromosome. Instead, an approach that uses an endogenous centromere that has never left a cell and that perpetuates the association with CENH3 would be needed. The introduction of telomere sequences into cells had been demonstrated in mammalian cells to generate small chromosomes with the arms cleaved away [17–19]. Thus, a similar approach was used in plants to bypass the epigenetic nature of centromeres [20,21](Fig. 1). The introduction of constructs containing plant telomere repeats at one end were introduced into maize embryos with a selectable marker either by Agrobacterium mediated or biolistically mediated transformation [21] (Fig. 1). In both cases, truncation of chromosomes was observed for a reasonable fraction of the transformation events. The details of the mechanism of truncation are not known but the end of the transgene carrying telomere sequences must trigger the attraction of the telomere cap that is then perpetuated through somatic embryogenesis and subsequent generations. The telomere repeat is identical in almost all plant species, with minor exception [22]. Therefore, telomere mediated truncation is predicted to work
Regardless of the means of generating minichromosomes, they have several properties that need to be addressed to maximize their utility. The production of numerous small chromosomes in maize has shown that, in general, they have reduced transmission through meiosis although their mitotic stability appears to be normal [31–33]. When multiple copies of very small chromosomes are present in meiosis, they are incapable of finding their partners and do not exhibit homologue pairing [31,33]. Furthermore, the sister chromatids of these small chromosomes tend to separate precociously at meiotic anaphase I instead of remaining adhered to each other until meiosis II, as do normal chromosomes [31,33]. Despite these transmission issues, it is conceivable that pollen selection genes could eventually be placed on minichromosomes such that only those grains containing the selection genes would be viable [6–8]. Examples might include the normal copy of nuclear male sterile genes on a minichromosome in a background of homozygous mutant or nuclear restorer genes of gametophytic cytoplasmic male sterility with the respective cytoplasm. These configurations would force the transmission of a single minichromosome with fidelity from one generation to the next. 2
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Fig. 1. Scheme for the production of engineered minichromosomes in maize using telomere-mediated chromosomal truncation. Telomere truncation utilizes truncation constructs, which contain plant telomere sequences, to truncate the targeted chromosome. 1A) A Ti (Tumor inducing) plasmid containing a truncation construct is transformed into a maize plant containing B chromosomes (indicated) using Agrobacterium transformation. 1B) A plasmid containing a truncation construct is coated onto gold particles, which are then bombarded into a maize young embryo or callus containing B chromosomes. 2) The truncation construct is delivered into the target maize tissue, where it can be transported to the nucleus. 3) Once in the nucleus, the truncation construct can insert into a double strand break on a B chromosome, which can result in the formation of a minichromosome (indicated).
transmission is of less concern for field propagation. In these cases, breeding programs develop a desired line that is then perpetuated vegetatively. Because the examples of minichromosomes that have been studied appear to have faithful mitotic behavior, this type of propagation would not lose the minichromosomes. Following the
1.4. Clonal crops For clonal crops that have breeding programs but vegetative propagation in the field (e.g. sugarcane, sweet potato, banana, poplar, rubber, potato, cacao, taro, palms, cassava et cetera), the issue of 3
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decades for the manipulation of transgenes in vivo particularly using site specific recombinases [39–41]. The tyrosine recombinases have been used to excise marker genes, to turn on or turn off gene expression and to mediate integration. The most used versions include Cre [40,42], FLP [43,44] and R recombinases [45]. Their mode of action uses target recombination sites that are identical but exhibit a directionality. By orienting the recombination sites in different directions, the intervening sequences can be flipped or excised as a circle. If an introduced circular DNA is present with a matching site in a chromosomal location, the circle can be incorporated via recombination catalyzed by the respective recombinase [46]. Because the tyrosine recombinases have identical target sites, the reaction can proceed in both directions. Mutant target sites have been used to favor the forward reaction [47]. An example of using Cre recombinase to remove a selectable marker from an engineered minichromosome has been published [30]. The other major class, serine recombinases, use different target sites for recombination. Thus, their reactions are irreversible in the absence of the corresponding excisionase. They are typically derived from bacterial/phage integration systems so the reacting sites are referred to as P (phage) and B (bacterial) attachment sequences. Recombination between P and B sites generate L and R sites that are not recognized by the solo recombinase, leading to irreversible reactions. In this class, phiC31 [48,49] and Bxb1 [50] have been shown to function in plant cells. A bioinformatic analysis distilled integrase candidates to increase the list to 34 that are not predicted to overlap in function [51]. Several candidates were shown to operate in bacterial cells. This collection of integrases has the potential to expand massively the manipulations of synthetic chromosomes. David Ow has proposed and shown to function in principle a stacking system that uses multiple selectable markers and recombinases to add new genes to pre-determined sites in chromosomes [52]. The stacked genes can be acted upon to remove selectable markers as the process continues to add new genes to the array [53]. This proof of concept could be applied to engineered minichromosomes to continue to add to them, which in this case would be an independent entity in the genome that would be separate from any linked genes in the standard genome. CRISPR-Cas9 technology could also be combined with the features described above to modify minichromosomes. The included genes could be edited to mutate unwanted expression or to delete undesirable features. To the extent that homologous recombination initiated by CRISPR-Cas9 can add short sequences to minichromosomes, this potential also exists. Another desirable future direction includes the development of a promoter library for the correct expression of foreign genes or endogenous genes for which one would want to alter their expression profile. Using the same promoter on multiple transgenes has the potential to induce gene silencing of the pair of transgenes so diversity of sequences is needed to avoid this complication. Synthetic promoters might eventually be developed but there is no substitute for the collection of promoters that have evolved over many eons to direct properly their cognate genes in the correct tissues and cells. As described above, the conditions to recover foundational minichromosomes and procedures to modify them in vivo have been established. The methodology has been demonstrated for means to add genes to target sequences so the ability to grow minichromosomes has also been established. As these techniques continue to be combined, the growth of minichromosomes will accelerate. If techniques can be developed to introduce successfully even larger fragments of DNA [54–56], then those procedures can be integrated into the assembly toolkit to grow synthetic chromosomes in leaps and bounds.
minichromosome in breeding programs can be conducted by simple PCR tracking. Useful traits could be stacked on a minichromosome with selection for the most favorable genetic background that would serve as the progenitor to bulk for distribution to the field. 1.5. Copy number manipulation In the case of engineered minichromosomes derived from the maize B chromosome, the potential exists to manipulate the copy number of the chromosomes. The B chromosome, as noted, is non-essential but is maintained in populations via a drive mechanism. This mechanism consists of nondisjunction at the second pollen mitosis, which makes the two maize sperm, followed by preferential fertilization of the egg by the sperm that contains the B chromosomes in the process of double fertilization [34,35]. The nondisjunction occurs at the centromere but there are also trans-acting factors on the long arm of the B chromosome that are required for this process to occur [36]. With minichromosomes that have the long arm removed, they are stabilized for their behavior at the second pollen mitosis because they are missing the trans-acting factors. However, if normal B chromosomes are added to the genotype to supply those factors, the minichromosomes can now undergo nondisjunction and can be amplified for copy number [32]. In a proof of concept experiment, the copy number of minichromosomes could be increased beyond 15 [32]. If an increased output is desired from an engineered mini, such a scheme could be used to achieve that goal. 1.6. Combining engineered minichromosomes with doubled haploid breeding For species that have a robust doubled haploid induction system, minichromosomes could be combined with this technology to transfer the engineered chromosome to new target lines [8]. Doubled haploid induction can occur by natural systems that cause the elimination of chromosomes from one parent after fertilization or by modifying CENH3 to do the same [37]. The haploids produced are then doubled to generate a progeny that is completely homozygous. This approach is widely used in industry to obtain new combinations of genes from hybrids and to short circuit an otherwise lengthy process of obtaining homozygous lines through inbreeding. Both these types of haploid generating systems sometimes have a retention of a chromosome from the parent whose chromosomes are usually eliminated [37,38]. The chromosomes that escape the elimination mechanism can then be maintained in the doubled haploid lines. If an engineered minichromosome were such a retained chromosomes, it would transfer all of its cargo to the doubled haploid, which would bypass extensive backcrossing schemes. The normal B chromosome of maize, as well other chromosomes, has been shown to be transferred into haploids when incorporated in haploid inducing lines [8,38]. While minichromosomes have reduced meiotic transmission, it seems likely that they could be recovering in this process as well. With high throughput identification methods, minichromosomes could be used to transfer included genes for novel traits to haploids that could be doubled to include the transgenes without introgression. It has also been suggested that this approach could be used to introduce editing machinery into haploids to bypass complications of bi-allelic edits and to perform massive scale genome editing [8]. 1.7. Building upon minichromosome platforms The constructs that are used for telomere mediated chromosomal truncation carry with them the selectable marker gene and potentially other cargo at the terminus of the newly minted chromosome. If only simple traits are desired, then no further modifications would be necessary. However, the impetus for the development of engineered minichromosome technology is the potential to stack multiple traits on a single chromosome entity and eventually to produce synthetic chromosomes. Proof of concept experiments have been described in the past
Funding Research on this topics in our laboratory is funded by National 4
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Science Foundation grant IOS-1339198.
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Engineered minichromosomes in plants James A. Birchler∗, Nathan C. Swyers Division of Biological Sciences, University of Missouri, 311 Tucker Hall, Columbia, MO, 65211-7400, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Artificial chromosomes Synthetic chromosomes Maize Telomere-mediated truncation Gene stacking Site specific recombinases
Artificial chromosome platforms are described in plants. Because the function of centromeres is largely epigenetic, attempts to produce artificial chromosomes with plant centromere DNA have failed. The removal of the centromeric sequences from the cell strips off the centromeric histone that is the apparent biochemical marker of centromere activity. Thus, engineered minichromosomes have been produced by telomere mediated chromosomal truncation. The introduction of telomere repeats will cleave the chromosome at the site of insertion and attach the accompanying transgenes in the process. Such truncation events have been documented in maize, Arabidopsis, barley, rice, Brassica and wheat. Truncation of the nonvital supernumerary B chromosome of maize is a favorite target but engineered minichromosomes derived from the normal A chromosomes have also been recovered. Transmission through mitosis of small chromosomes is apparently normal but there is loss during meiosis. Potential solutions to address this issue are discussed. With procedures now well established to produce the foundation for artificial chromosomes in plants, current efforts are directed at building them up to specification using gene stacking methods and editing techniques.
1. Introduction In the past several decades the amount of arable land on the planet has been decreasing while the human population has been increasing. If the human population wishes to improve its standard of living, or indeed to simply maintain it, agriculture will need a revolution in order to produce more food in those coming decades than has ever been produced before [1]. Agriculture has had a profound impact on the biosphere with a predicted loss of natural animal species consisting of one out of 8 of them in the coming decades [2]. Ideas to adapt to these changes include the use of synthetic biology utilizing the directed evolution of enzymatic functions and synthetic transcription factors to produce plants with new combinations of properties. Various efforts are presently in place to attempt to confer nitrogen fixation to the major cereal crops that provide the vast majority of calories to the world but at the expense of considerable fertilizer application. Other possibilities are programs to improve the efficiency of photosynthesis, to confer the efficient C4 photosynthesis to species with C3 such as rice, to sequester carbon dioxide in plants, to enhance the nutritional components of crops, to modify crops to withstand environmental challenges and to provide plants with resistances to insect, viral, bacterial and fungal pests. Through millennia, humans have modified plants via unwitting and intentional selection to bring civilization to its current state. Such activity must continue.
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Synthetic approaches coupled with natural variation, which must also be secured, promise to be an effective avenue for crop improvement. One contribution to such an effort would be the development of means to construct independent chromosomes, which might harbor a collection of plant modifications. Artificial chromosomes were first assembled in yeast [3] and was the paradigm for applications in other species. They were generated by the assembly of centromeres, telomeres, an origin of replication and a selectable marker. When reintroduced into yeast cells, they functioned autonomously. However, using that paradigm for plants has not been successful to date despite numerous attempts in various species. The transformation of DNA from known centromere regions of chromosomes only produces stable integrations in the most rigorously controlled experiments [4]. The apparent reason is that the function of centromeres in plants is basically epigenetic with the underlying DNA not being responsible for their activity [5]. Instead, centromere function appears to rely on the presence of the centromere specific variant of histone H3, whose location on the chromosome is perpetuated from one cell cycle to the next. The role of engineered minichromosomes/artificial chromosomes/ synthetic chromosomes in plants has garnered attention in the past few years for a variety of reasons [6–8]. Not only do they have potential as a facile experimental tool but they hold promise for a variety of practical applications. Current methodologies for plant transformation rely on random integrations into the genome that result in variable expression
Corresponding author. E-mail address: [email protected] (J.A. Birchler).
https://doi.org/10.1016/j.yexcr.2020.111852 Received 3 November 2019; Accepted 14 January 2020 0014-4827/ © 2020 Elsevier Inc. All rights reserved.
Please cite this article as: James A. Birchler and Nathan C. Swyers, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2020.111852
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in basically all genetic models and crop species. Indeed, in addition to maize, telomere mediated truncation has been reported in Arabidopsis [23,24], barley [25], rice [26], wheat [27], and Brassica [28]. When normal chromosomes are truncated an aneuploid condition is generated that might be detrimental to plant growth and vigor and any deletions of vital genes will abort the haploid gametophyte generation. Despite these considerations, various approaches can be used to produce minichromosomes and recover them. In maize, there is a supernumerary B chromosome that is nonvital [29]; thus, it is a convenient target for truncation [21]. A strategy is outlined in Fig. 1 for utilizing telomere-mediated chromosomal truncation to target a B chromosome in maize to create an engineered minichromosomes. Another approaches that have been used is to target a tetraploid as the starting material [26] that would diminish any aneuploid effect and cover any deficiencies in the gametophyte generation, which in this case would be diploid. An additional approach was to use a telotrisomic genotype of rice that contains an extra chromosome arm as the trisomic addition. Truncation of this chromosome would actually remove aneuploid effects and produced an engineered minichromosome in the process. Wheat is a natural allohexaploid that can tolerate nullisomic conditions so truncation of chromosomes can be recovered [27]. Also, the rye B chromosome has been transferred to wheat and could serve as a target. Despite these aids in recovering truncated chromosomes, it is possible to obtain them from diploid targets directly. In addition to adding telomere repeats to selectable marker constructs for transformation, one can simply perform a cobombardment of the marker cassette with free telomere repeats [30]. An example was recovered that removed both arms of a chromosome but included the addition of the marker cassette. Presumably, the removal of both arms occurred to create a functional monosomic condition but the doubling of the remaining normal homologue occurred during the tissue culture selection. This example illustrates that even using a diploid target it is possible to recover engineered minichromosomes. The recovery of small chromosomes to date has relied on the random integration of the truncating constructs. This approach has been sufficiently frequent to recover very small chromosomes with added genes. However, in the current era of extensive genome sequence data and the ability to edit specific sequences, it is conceivable that targeted truncation might be possible in the future. One can imagine that it could be possible to remove all of the endogenous genes from a chromosome leaving the centromere and added DNA to build the chromosome to specification. Such an approach would leave a natural centromere that has never left a plant cell to maintain its function but would not leave any genes in the minichromosome foundation.
states of the transgenes and are potentially mutagenic if they impact endogenous gene expression. An independent chromosome would avoid these effects as well as any complications of linkage to undesired endogenous genes when incorporating transgenes from one variety to another. Also, combining multiple unlinked transgenes for stacking traits quickly becomes quite complicated as the assembly of unlinked insertions builds. Autonomously built chromosomes from scratch would overcome these issues. 1.1. Epigenetic nature of centromeres Before proceeding to a description of producing engineered minichromosome in plants, a discussion of centromere functions is needed. The realization of the frequency with which centromeres in plants could become inactive came from experiments that generated functionally dicentric chromosomes repeatedly over the life cycle of maize [9]. Among the progeny of plants with this configuration were sequence dicentrics with only a single centromere with retention of the centromere specific centromeric H3 (CENH3). Once this realization became evident, many additional examples of inactive centromeres were realized [10,11]. Directed inactivation was achieved by tying together, via crossing over with a unique foldback chromosome, a normal large and a version of the same centromere that had been experimentally reduced in size [10,12]. Recombination to produce a tug of war resulted in the smaller centromere becoming inactive with loss of CENH3. The collective results of many examples of inactive centromeres indicates the inactivation can occur over a maximum of one or a few cell cycles and is perpetuated in the inactive state in perpetuity. The flip side observation indicating an epigenetic feature of plant centromeres is the repeated finding of de novo formed centromeres on chromatin fragments that are missing canonical centromeric DNA [13–15]. ChIP-Seq experiments using antibodies against the respective CENH3 performed on fragments with known origins confirmed that new centromere formation had occurred over unique sequences present in chromosome arms from which the fragment originated [14,15]. Numerous cases of de novo centromeres have now been determined and, in some cases, the timeframe of formation of the fragments had been documented, which demonstrated in these cases as well that they emerged over only one or a few cell cycles and then perpetuated over decades. Variation in the exact position of CENH3 in centromeric regions in different varieties of maize indicates that centromere inactivation and de novo formation is a natural occurrence on a regular basis [16]. 1.2. Telomere mediated chromosomal truncation
1.3. Behavior of small chromosomes in meiosis With the realization of the epigenetic nature of centromeres in plants, it became evident that the removal of centromeric DNA from cells and its reintroduction would be ineffective as a component of an artificial chromosome. Instead, an approach that uses an endogenous centromere that has never left a cell and that perpetuates the association with CENH3 would be needed. The introduction of telomere sequences into cells had been demonstrated in mammalian cells to generate small chromosomes with the arms cleaved away [17–19]. Thus, a similar approach was used in plants to bypass the epigenetic nature of centromeres [20,21](Fig. 1). The introduction of constructs containing plant telomere repeats at one end were introduced into maize embryos with a selectable marker either by Agrobacterium mediated or biolistically mediated transformation [21] (Fig. 1). In both cases, truncation of chromosomes was observed for a reasonable fraction of the transformation events. The details of the mechanism of truncation are not known but the end of the transgene carrying telomere sequences must trigger the attraction of the telomere cap that is then perpetuated through somatic embryogenesis and subsequent generations. The telomere repeat is identical in almost all plant species, with minor exception [22]. Therefore, telomere mediated truncation is predicted to work
Regardless of the means of generating minichromosomes, they have several properties that need to be addressed to maximize their utility. The production of numerous small chromosomes in maize has shown that, in general, they have reduced transmission through meiosis although their mitotic stability appears to be normal [31–33]. When multiple copies of very small chromosomes are present in meiosis, they are incapable of finding their partners and do not exhibit homologue pairing [31,33]. Furthermore, the sister chromatids of these small chromosomes tend to separate precociously at meiotic anaphase I instead of remaining adhered to each other until meiosis II, as do normal chromosomes [31,33]. Despite these transmission issues, it is conceivable that pollen selection genes could eventually be placed on minichromosomes such that only those grains containing the selection genes would be viable [6–8]. Examples might include the normal copy of nuclear male sterile genes on a minichromosome in a background of homozygous mutant or nuclear restorer genes of gametophytic cytoplasmic male sterility with the respective cytoplasm. These configurations would force the transmission of a single minichromosome with fidelity from one generation to the next. 2
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Fig. 1. Scheme for the production of engineered minichromosomes in maize using telomere-mediated chromosomal truncation. Telomere truncation utilizes truncation constructs, which contain plant telomere sequences, to truncate the targeted chromosome. 1A) A Ti (Tumor inducing) plasmid containing a truncation construct is transformed into a maize plant containing B chromosomes (indicated) using Agrobacterium transformation. 1B) A plasmid containing a truncation construct is coated onto gold particles, which are then bombarded into a maize young embryo or callus containing B chromosomes. 2) The truncation construct is delivered into the target maize tissue, where it can be transported to the nucleus. 3) Once in the nucleus, the truncation construct can insert into a double strand break on a B chromosome, which can result in the formation of a minichromosome (indicated).
transmission is of less concern for field propagation. In these cases, breeding programs develop a desired line that is then perpetuated vegetatively. Because the examples of minichromosomes that have been studied appear to have faithful mitotic behavior, this type of propagation would not lose the minichromosomes. Following the
1.4. Clonal crops For clonal crops that have breeding programs but vegetative propagation in the field (e.g. sugarcane, sweet potato, banana, poplar, rubber, potato, cacao, taro, palms, cassava et cetera), the issue of 3
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decades for the manipulation of transgenes in vivo particularly using site specific recombinases [39–41]. The tyrosine recombinases have been used to excise marker genes, to turn on or turn off gene expression and to mediate integration. The most used versions include Cre [40,42], FLP [43,44] and R recombinases [45]. Their mode of action uses target recombination sites that are identical but exhibit a directionality. By orienting the recombination sites in different directions, the intervening sequences can be flipped or excised as a circle. If an introduced circular DNA is present with a matching site in a chromosomal location, the circle can be incorporated via recombination catalyzed by the respective recombinase [46]. Because the tyrosine recombinases have identical target sites, the reaction can proceed in both directions. Mutant target sites have been used to favor the forward reaction [47]. An example of using Cre recombinase to remove a selectable marker from an engineered minichromosome has been published [30]. The other major class, serine recombinases, use different target sites for recombination. Thus, their reactions are irreversible in the absence of the corresponding excisionase. They are typically derived from bacterial/phage integration systems so the reacting sites are referred to as P (phage) and B (bacterial) attachment sequences. Recombination between P and B sites generate L and R sites that are not recognized by the solo recombinase, leading to irreversible reactions. In this class, phiC31 [48,49] and Bxb1 [50] have been shown to function in plant cells. A bioinformatic analysis distilled integrase candidates to increase the list to 34 that are not predicted to overlap in function [51]. Several candidates were shown to operate in bacterial cells. This collection of integrases has the potential to expand massively the manipulations of synthetic chromosomes. David Ow has proposed and shown to function in principle a stacking system that uses multiple selectable markers and recombinases to add new genes to pre-determined sites in chromosomes [52]. The stacked genes can be acted upon to remove selectable markers as the process continues to add new genes to the array [53]. This proof of concept could be applied to engineered minichromosomes to continue to add to them, which in this case would be an independent entity in the genome that would be separate from any linked genes in the standard genome. CRISPR-Cas9 technology could also be combined with the features described above to modify minichromosomes. The included genes could be edited to mutate unwanted expression or to delete undesirable features. To the extent that homologous recombination initiated by CRISPR-Cas9 can add short sequences to minichromosomes, this potential also exists. Another desirable future direction includes the development of a promoter library for the correct expression of foreign genes or endogenous genes for which one would want to alter their expression profile. Using the same promoter on multiple transgenes has the potential to induce gene silencing of the pair of transgenes so diversity of sequences is needed to avoid this complication. Synthetic promoters might eventually be developed but there is no substitute for the collection of promoters that have evolved over many eons to direct properly their cognate genes in the correct tissues and cells. As described above, the conditions to recover foundational minichromosomes and procedures to modify them in vivo have been established. The methodology has been demonstrated for means to add genes to target sequences so the ability to grow minichromosomes has also been established. As these techniques continue to be combined, the growth of minichromosomes will accelerate. If techniques can be developed to introduce successfully even larger fragments of DNA [54–56], then those procedures can be integrated into the assembly toolkit to grow synthetic chromosomes in leaps and bounds.
minichromosome in breeding programs can be conducted by simple PCR tracking. Useful traits could be stacked on a minichromosome with selection for the most favorable genetic background that would serve as the progenitor to bulk for distribution to the field. 1.5. Copy number manipulation In the case of engineered minichromosomes derived from the maize B chromosome, the potential exists to manipulate the copy number of the chromosomes. The B chromosome, as noted, is non-essential but is maintained in populations via a drive mechanism. This mechanism consists of nondisjunction at the second pollen mitosis, which makes the two maize sperm, followed by preferential fertilization of the egg by the sperm that contains the B chromosomes in the process of double fertilization [34,35]. The nondisjunction occurs at the centromere but there are also trans-acting factors on the long arm of the B chromosome that are required for this process to occur [36]. With minichromosomes that have the long arm removed, they are stabilized for their behavior at the second pollen mitosis because they are missing the trans-acting factors. However, if normal B chromosomes are added to the genotype to supply those factors, the minichromosomes can now undergo nondisjunction and can be amplified for copy number [32]. In a proof of concept experiment, the copy number of minichromosomes could be increased beyond 15 [32]. If an increased output is desired from an engineered mini, such a scheme could be used to achieve that goal. 1.6. Combining engineered minichromosomes with doubled haploid breeding For species that have a robust doubled haploid induction system, minichromosomes could be combined with this technology to transfer the engineered chromosome to new target lines [8]. Doubled haploid induction can occur by natural systems that cause the elimination of chromosomes from one parent after fertilization or by modifying CENH3 to do the same [37]. The haploids produced are then doubled to generate a progeny that is completely homozygous. This approach is widely used in industry to obtain new combinations of genes from hybrids and to short circuit an otherwise lengthy process of obtaining homozygous lines through inbreeding. Both these types of haploid generating systems sometimes have a retention of a chromosome from the parent whose chromosomes are usually eliminated [37,38]. The chromosomes that escape the elimination mechanism can then be maintained in the doubled haploid lines. If an engineered minichromosome were such a retained chromosomes, it would transfer all of its cargo to the doubled haploid, which would bypass extensive backcrossing schemes. The normal B chromosome of maize, as well other chromosomes, has been shown to be transferred into haploids when incorporated in haploid inducing lines [8,38]. While minichromosomes have reduced meiotic transmission, it seems likely that they could be recovering in this process as well. With high throughput identification methods, minichromosomes could be used to transfer included genes for novel traits to haploids that could be doubled to include the transgenes without introgression. It has also been suggested that this approach could be used to introduce editing machinery into haploids to bypass complications of bi-allelic edits and to perform massive scale genome editing [8]. 1.7. Building upon minichromosome platforms The constructs that are used for telomere mediated chromosomal truncation carry with them the selectable marker gene and potentially other cargo at the terminus of the newly minted chromosome. If only simple traits are desired, then no further modifications would be necessary. However, the impetus for the development of engineered minichromosome technology is the potential to stack multiple traits on a single chromosome entity and eventually to produce synthetic chromosomes. Proof of concept experiments have been described in the past
Funding Research on this topics in our laboratory is funded by National 4
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Science Foundation grant IOS-1339198.
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