Chromosome Elimination Research Articles

Using CRISPR-Cas9 In Human Fetuses to Prevent Trisomy 16 and Trisomy 22 Induced Miscarriages

About 50% of abortions have been found to be caused by aneuploidy, roughly 60% of which are trisomy. The most common trisomy occurs on chromosome 16, 21, and 22. However, trisomy 21 (also called Down Syndrome) is viable in about 57% of cases, while trisomy 16 and 22 result in miscarriage in nearly every pregnancy. Thus, additional therapies and treatments must be explored, especially through quickly advancing techniques such as gene editing. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) combined with the cleaving endonuclease CRISPR-associated protein (Cas9) harbors potential for targeted chromosome deletion, possibly increasing the chance of survival in fetuses with trisomy 16 or 22. However, the risks and safety benefits during genetic diagnosing and potential treatment of the fetus should also be considered. Successfully demonstrated approaches of editing or eliminating extraneous chromosomes in studies of both animal models and lab-cultured human embryos will be discussed. In particular, this paper will examine possible gene editing approaches such as elimination of entire chromosomes, large-scale deletions, and chromosomal truncations to target trisomy. In order to determine the efficacy of these approaches in trisomy, the use of CRISPR-Cas9 to specifically treat trisomy via autosomal deletion and counter-selection markers will be deliberated. Delivery methods for the in-utero therapy includes viral techniques such as retroviruses and adeno-associated vectors (AAVs) as well as non-viral techniques like electroporation and nanoparticles. This paper will propose hypothetical treatments for CRISPR-Cas9 in human embryos to target trisomy 16 and 22 while also examining the bioethical implications of doing so.

Cytogenetic Effects in Patients after Computed Tomography Examination.

Millions of people around the world are exposed to low doses of ionizing radiation from diagnostic computed tomography (CT) scans. Currently available data on the potential cancer risk after CT scans are contradictory and therefore demand further investigations. The aim of the current study was to obtain estimations of genome damage after CT scans in 42 non-cancer patients and to conduct a comparison of the results with 22 control subjects. The frequency of dicentric ring chromosomes and chromosome breaks was significantly increased in irradiated patients compared to the controls. The distribution of dicentrics among the cells demonstrated non-Poisson distribution that reflected non-uniform and partial-body radiation exposure. A fraction of patients followed Poisson distribution, which is typical for uniform whole-body exposures. Some patients demonstrated a level of dicentrics similar to the control subjects. The individual variations in the frequency and dicentric distribution suggested complex mechanisms of chromosome aberration induction and elimination that could be associated with individual radiosensitivity, as well as previous diagnostics that used ionizing radiation or the redistribution of small fractions of irradiated lymphocytes within the circulatory pull. In conclusion, CT scans may cause genome damage and possible increases in cancer risk. The introduction of a specific follow-up of such patients, especially in the case of repeated CT scans, is suggested.

Direct Transfer and Consolidation of Synthetic Yeast Chromosomes by Abortive Mating and Chromosome Elimination.

Large DNA transfer technology has been challenged with the rapid development of large DNA assembly technology. The research and application of synthetic yeast chromosomes have been mostly limited in the assembled host itself. The mutant of KAR1 prevents nuclear fusion during yeast mating, and occasionally single chromosome can be transferred from one parental nucleus to another. Using the kar1 mutant method, four synthetic yeast chromosomes of Sc2.0 (synIII, synV, synX, synXII) were transferred to wild-type yeasts separately. SynIII was also transferred into an industrial strain Y12, resulting in an improvement of thermotolerance. Moreover, by combining abortive mating and chromosome elimination by CRISPR-Cas9, which has been reported in our previous study, we developed a strategy for consolidation of multiple synthetic yeast chromosomes. Compared to the previous pyramidal strategy using endoreduplication backcross, our method is a linear process independent of meiosis, providing a convenient path for accelerating consolidation of Sc2.0 chromosomes. Overall, the method of transfer and consolidation of synthetic yeast chromosomes by abortive mating and chromosome elimination enables a novel route that large DNA was assembled in donor yeast and then in vivo directly transferred to receptor yeasts, enriching the manipulation tools for synthetic genomics.

Development of Transformation for Genome Editing of an Emerging Model Organism.

With the advances in genomic sequencing, many organisms with novel biological properties are ripe for use as emerging model organisms. However, to make full use of them, transformation methods need to be developed to permit genome editing. Here, we present the development of transformation for the fungus fly Bradysia (Sciara) coprophila; this may serve as a paradigm for the development of transformation for other emerging systems, especially insects. Bradysia (Sciara) has a variety of unique biological features, including locus-specific developmentally regulated DNA amplification, chromosome imprinting, a monopolar spindle in male meiosis I, non-disjunction of the X chromosome in male meiosis II, X chromosome elimination in early embryogenesis, germ-line-limited (L) chromosomes and high resistance to radiation. Mining the unique biology of Bradysia (Sciara) requires a transformation system to test mutations of DNA sequences that may play roles for these features. We describe a Bradysia (Sciara) transformation system using a modified piggyBac transformation vector and detailed protocols we have developed to accommodate Bradysia (Sciara) specific requirements. This advance will provide a platform for us and others in the growing Bradysia (Sciara) community to take advantage of this unique biological system. In addition, the versatile piggyBac vectors described here and transformation methods will be useful for other emerging model systems.

Experimental method for haplotype phasing across the entire length of chromosome 21 in trisomy 21 cells using a chromosome elimination technique

Modern sequencing technologies produce a single consensus sequence without distinguishing between homologous chromosomes. Haplotype phasing solves this limitation by identifying alleles on the maternal and paternal chromosomes. This information is critical for understanding gene expression models in genetic disease research. Furthermore, the haplotype phasing of three homologous chromosomes in trisomy cells is more complicated than that in disomy cells. In this study, we attempted the accurate and complete haplotype phasing of chromosome 21 in trisomy 21 cells. To separate homologs, we established three corrected disomy cell lines (ΔPaternal chromosome, ΔMaternal chromosome 1, and ΔMaternal chromosome 2) from trisomy 21 induced pluripotent stem cells by eliminating one chromosome 21 utilizing the Cre-loxP system. These cells were then whole-genome sequenced by a next-generation sequencer. By simply comparing the base information of the whole-genome sequence data at the same position between each corrected disomy cell line, we determined the base on the eliminated chromosome and performed phasing. We phased 51,596 single nucleotide polymorphisms (SNPs) on chromosome 21, randomly selected seven SNPs spanning the entire length of the chromosome, and confirmed that there was no contradiction by direct sequencing.

B-A Chromosome Translocations Possessing an A Centromere Partly Overcome the Root-Restricted Process of Chromosome Elimination in Aegilops speltoides.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from the programmed elimination of chromosomes. Aegilops speltoides is an annual diploid species from the Poaceae family, with a maximum number of eight B chromosomes (Bs) in addition to its inherent seven pairs of standard A chromosomes (As). The Bs of this species undergo precise elimination in roots early in embryo development. In areal parts of the plant, the number of Bs is stable. To affect the root restricted process of B chromosome elimination, we employed X-ray mutagenesis, and different types of restructured Bs were identified. Standard Bs were observed in all analyzed shoots of mutagenized plants, while B-A translocations were only observed in 35.7% of F1 plants. In total 40 different B variants inconsistently escaped the elimination process in roots. As a result, mosaicism of B chromosome variants was found in roots. Only a small B chromosome fragment fused to an A chromosome was stably maintained in roots and shoots across F1 to F3 generations. The absence of B-A translocation chromosomes possessing a derived B centromere in root cells implies that the centromere of the B is a key component of the chromosome elimination process.

Triticale haploidy <i>in vitro</i> (literature review)

Triticale (× Triticosecale Wittmack) is a hybridized grain crop developed from wheat and rye crossings. Today, triticale is a multipurpose commercial grain crop with great potential as a human food and animal feed. The sown area of the new grain crop in the world reached 4 million hectares in 2018, grain production was about 14 million tons. The current climate change, the rapid evolution of pathogens, as well as the requirements of the modern market dictate the necessity for accelerated development of varieties while reducing the cost of their development. The production of double haploids makes it possible to reduce the time required for the development of homozygous lines by an average of 5–7 years. For the mass production of haploid triticale plants in vitro, there are used two methods, namely anther/microspore culture and distant hybridization followed by selective chromosome elimination of the pollinator. The most critical factors for the success of developing haploids in anther culture are a genotype, growing conditions of donor plants, a microspore development stage, stress effects on heads or anthers, and a nutrient media. Among the unresolved problems of the method are a genotypic dependence, a high incidence of albinism and a presence of aneuploids in the androgenic plant progeny. The rye genome is more often involved in chromosomal transformations than the wheat genome. Most aneuploids are nullisomics, most often on the 2R and 5R chromosomes. Nullisomic plants for 2R and 5R chromosomes have fewer number of spikelets per head and fewer number of kernels per head. In order to develop haploids by the method of selective chromosome elimination during distant hybridization, there have been successfully used such grain crops whose pollen is insensitive to Kr-genes, as maize (Zea mays L.) and wild cereal grass ‘Imperata Cylindrical’ (Imperata cylindrical L.). The advantages of the method are less genotypic dependence, absence of albino plants, genetic stability of regenerants, and reduced costs for developing haploid plants. The length of flowering period of ‘Imperata Cylindrical’ and the absence of the need to combine the timing of flowering period of the parents ensure the economy and efficiency of using this species when developing haploid triticale plants. The purpose of the current review was to characterize the methods of mass development of haploid triticale plants, to describe their advantages and disadvantages when being used in the breeding process.

Matrilineal empowers wheat pollen with haploid induction potency by triggering postmitosis reactive oxygen species activity.

Reactive oxygen species (ROS) play important roles during anther and pollen development. DNA damage may cause chromosome fragmentation that is considered to underlie chromosome elimination for haploid induction by matrilineal pollen, a key step in MATRILINEAL-based double haploid breeding technology. But when and how DNA damage occurs is unknown. We performed comparative studies of wheat pollens from the wild-type and the CRISPR/Cas9 edited matrilineal mutant (mMTL). Chemical assays detected a second wave of ROS in mMTL pollen at the three-nuclei-stage and subsequently, along with reduced antioxidant enzyme activities. RNA-seq analysis revealed disturbed expression of genes for fatty acid biosynthesis and ROS homoeostasis. Gas chromatography-mass spectrometry measurement identified abnormal fatty acid metabolism that may contribute to defective mMTL pollen walls as observed using electron microscopy, consistent with the function of MTL as a phospholipase. Moreover, DNA damage was identified using TdT-mediated dUTP nick-end labelling and quantified using comet assays. Velocity patterns showed that ROS increments preceded that of DNA damage over the course of pollen maturation. Our work hypothesises that mMTL-triggered later-stage-specific ROS causes DNA damage that may contribute to chromosome fragmentation and hence chromosome elimination during haploid induction. These findings may provide more ways to accelerate double haploid-based plant breeding.

In vivo haploid induction potential of Himalayan maize (Zea mays) and cogon grass (Imperata cylindrica) gene pools in different segregational cycles of intra and inter-generic crosses of wheat

AbstractMaize and Imperata cylindrica have been utilized globally as a pollen source for induction of haploids in wheat through chromosome elimination technique. Pollen parents with a higher haploid induction rate are desired for recovering the high frequency of haploids in wheat and related species. The present investigation was carried out with the aim to assess haploid induction efficiency of diverse germplasm of maize and I. cylindrica in different generations of intra and intergeneric crosses of hexaploid and tetraploid wheat and triticale-wheat derivatives. Crosses of twenty-six lines (female) with each of two I. cylindrica and twenty-one maize genotypes (testers) were evaluated for four haploid induction parameters viz., pseudoseed formation frequency (PFF), embryo formation frequency (EFF), haploid regeneration frequency (HRF) and haploid formation frequency (HFF). I. cylindrica outperformed maize in haploid induction rate with a frequency of embryos formed with I. cylindrica (18.39%) were significantly higher as compared to maize (4.08%). In the case of I. cylindrica genotype Ic-ye identified best with mean EFF of 30.55, 14.48 and 25.43% for hexaploids, tetraploids and triticale × wheat derivatives, respectively whereas in the case of maize genotype HPMC-60 performed best with EFF of 12.61% for hexaploids, HPMC-58 (12.58%) for tetraploids and HPMC-16 for triticale × wheat derivatives with EFF of 8.91%. I. cylindrica genotype Ic-ye and maize genotypes HPMC-14, HPMC-53, HPMC-60, HPMC-64 with significantly positive GCA for haploid induction parameters may be utilized as efficient pollen parents for recovering higher frequency of haploids in wheat.

Delete and survive: strategies of programmed genetic material elimination in eukaryotes.

ABSTRACTGenome stability is a crucial feature of eukaryotic organisms because its alteration drastically affects the normal development and survival of cells and the organism as a whole. Nevertheless, some organisms can selectively eliminate part of their genomes from certain cell types during specific stages of ontogenesis. This review aims to describe the phenomenon of programmed DNA elimination, which includes chromatin diminution (together with programmed genome rearrangement or DNA rearrangements), B and sex chromosome elimination, paternal genome elimination, parasitically induced genome elimination, and genome elimination in animal and plant hybrids. During programmed DNA elimination, individual chromosomal fragments, whole chromosomes, and even entire parental genomes can be selectively removed. Programmed DNA elimination occurs independently in different organisms, ranging from ciliate protozoa to mammals. Depending on the sequences destined for exclusion, programmed DNA elimination may serve as a radical mechanism of dosage compensation and inactivation of unnecessary or dangerous genetic entities. In hybrids, genome elimination results from competition between parental genomes. Despite the different consequences of DNA elimination, all genetic material destined for elimination must be first recognised, epigenetically marked, separated, and then removed and degraded.

Preventing aneuploidy: The groom must wait until the bride is ready.

Fertilization often triggers the final step of haploidization of the female gamete genome. In this issue, Mori et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202012001) identify two successive actin-dependent mechanisms that delay fusion of maternal and paternal chromosomes, preventing inadvertent elimination of paternal chromosomes together with maternal ones.

High contiguity de novo genome assembly and DNA modification analyses for the fungus fly, Sciara coprophila, using single-molecule sequencing

BackgroundThe lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features.ResultsWe assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage.ConclusionsWe determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.

RanGTP and the actin cytoskeleton keep paternal and maternal chromosomes apart during fertilization.

Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg-sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg-sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg-sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.

Cytogenetic events in the endosperm of amphiploid Avena magna\u2009\xd7\u2009A. longiglumis

This study analysed cytogenetic events occurring in the syncytial endosperm of the Avena magna H. C. Murphy & Terrell × Avena longiglumis Durieu amphiploid, which is a product of two wild species having different genomes. Selection through the elimination of chromosomes and their fragments, including those translocated, decreased the level of ploidy in the endosperm below the expected 3n, leading to the modal number close to 2n. During intergenomic translocations, fragments of the heterochromatin-rich C-genome were transferred to the D and Al genomes. Terminal and non-reciprocal exchanges dominated, whereas other types of translocations, including microexchanges, were less common. Using two probes and by counterstaining with DAPI, the A. longiglumis and the rare exchanges between the D and Al genomes were detected by GISH. The large discontinuity in the probe labelling in the C chromosomes demonstrated inequality in the distribution of repetitive sequences along the chromosome and probable intragenomic rearrangements. In the nucleus, the spatial arrangement of genomes was non-random and showed a sectorial-concentric pattern, which can vary during the cell cycle, especially in the less stable tissue like the hybrid endosperm.

Potential, Challenges and Strategies Involved in Gene Introgression from Wild Relatives of Vegetable Crops: A Review

Wide hybridization is an important plant breeding method as it helps in broadening the gene pool of a crop when the desired variation is not sufficient or absent within the same gene pool. Wild genetic resources are the potential source of desirable genes for various characters of crop plants. It plays a significant role in transferring traits of interest like disease and insect resistance, improved quality, earliness, dwarfness, increased yield, abiotic stress tolerance and manipulation in mode of reproduction etc. in crop plants. It has been instrumental in transferring disease resistance from wild species into many vegetable crops. For example, resistance to late blight, leaf roll and potato virus X, yellow vein mosaic virus and powdery mildew in okra, bacterial wilt, tomato leaf curl virus, early and late blight in tomato has been transferred from wild species into cultivated species. Wild species has been used to improve the quality traits of some crops like carotenoid content in carrot and tomato, starch content in potato etc. Sometimes distant hybridization leads to creation of entirely new crop species. Chromosome elimination technique followed by wide hybridization has been successfully used in the production of double haploids in some crops. However there are some lacunae in wide hybridization such as cross incompatibility, hybrid inviability, hybrid sterility and hybrid breakdown. Some special techniques viz. ploidy manipulation, pistil manipulation, growth regulators treatments, bridge crossing, grafting and embryo rescue have to be adopted to make distant hybridization successful in such cases.

Back-cross introgression of ‘Tour’ cytoplasm from Brassica napus through in vitro embryo rescue reveals partial restoration of sterility in B. oleracea

Cytoplasmic male sterility (CMS) is the most commonly used mechanisms for developing F1 hybrids in Brassica oleracea. Ogura cytoplasm is the only source of CMS system used widely in B. oleracea and diversification of CMS system is needed to avoid any imminent epidemic. We have developed sesquidiploids and backcross progenies involving the cybrid, B. napus with B. tournefortii (Tour) cytoplasm and B. oleracea for development of novel CMS system through different approaches of in vitro embryo rescue. Maturity of the ovaries and ovules were found to play important role in recovery of inter-specific hybrids through embryo rescue. The male sterility conferred by ‘Tour’ cytoplasm was partially restored by Rf genes in the B. oleracea nuclear genome and identification of perfect maintainer is necessary in developing ‘Tour’ based CMS system. The analysis of the sesquidiploid F1 hybrids and back-cross progenies revealed frequent occurrence of A and C sub-genome aneuploids and euploids. Chromosome elimination and fragmentation of the A and C genome specific linkage groups indicated the role of autosysndesis and allosyndesis in the AnCnCo sesquidiploid. The dominance of the B. napus like gametes was also evident in the first backcross progenies. The developed back-cross population with ‘Tour’ cytoplasm will be useful in developing stable CMS system in B. oleracea as an alternative to the available Ogura based lines for commercial hybrid breeding programme.

Uniparental Genome Elimination in Australian Carp Gudgeons.

Metazoans usually reproduce sexually, blending the unique identity of parental genomes for the next generation through functional crossing-over and recombination in meiosis. However, some metazoan lineages have evolved reproductive systems where offspring are either full (clonal) or partial (hemiclonal) genetic replicas. In the latter group, the process of uniparental genome elimination selectively eliminates either the maternal or paternal genome from germ cells, and only one parental genome is selected for transmission. Although fairly common in plants, hybridogenesis (i.e., clonal haploidization via chromosome elimination) remains a poorly understood process in animals. Here, we explore the proximal cytogenomic mechanisms of somatic and germ cell chromosomes in sexual and hybrid genotypes of Australian carp gudgeons (Hypseleotris) by tracing the fate of each set during mitosis (in somatic tissues) and meiosis (in gonads). Our comparative study of diploid hybrid and sexual individuals revealed visually functional gonads in male and female hybrid genotypes and generally high karyotype variability, although the number of chromosome arms remains constant. Our results delivered direct evidence for classic hybridogenesis as a reproductive mode in carp gudgeons. Two parental sets with integral structure in the hybrid soma (the F1 constitution) contrasted with uniparental chromosomal inheritance detected in gonads. The inheritance mode happens through premeiotic genome duplication of the parental genome to be transmitted, whereas the second parental genome is likely gradually eliminated already in juvenile individuals. The role of metacentric chromosomes in hybrid evolution is also discussed.

Optimization of chromosome doubling treatment for efficient in vivo doubled haploid production in maize

Abstract An investigation to optimize the protocol for application of colchicine for enhancing the doubled haploid production in maize was done. 106 maize genotypes were used as maternal parents, whereas, pollen source involved tropically adopted haploid inducer (TAIL P1 and TAIL hybrid). After the elimination of chromosomes of inducer lines, haploid seeds were obtained from the crosses. Haploid seedlings were treated with three different doses, such as 0.04, 0.06 and 0.08 per cent of colchicines for different durations (8, 12 and 15 hours). The response of various colchicine concentrations applied for different time durations revealed significant differences at P ≤ 0.05 for various parameters viz., per cent plants survivability, stalk colour, the fertility of tassel, silk present/absent, pollen viability, seed set and per cent doubled haploid formation. In maize, colchicine doses of 0.04 per cent for 12 hours and 0.06 per cent for 8 hours, respectively were established as optimum for enhanced doubled haploid production. But among these two, 0.04 per cent for 12 hours was observed to be best dose for doubled haploid production in maize.

Centromere Engineering as an Emerging Tool for Haploid Plant Production: Advances and Challenges.

Haploid production is of great importance in plant breeding programs. Doubled haploid technology accelerates the generation of inbred lines with homozygosity in all loci in a single year. Haploids can be induced in vitro via cultivating the haploid gametes or in vivo through inter- and intraspecific hybridization. Haploid induction through centromere engineering is a novel system that is theoretically applicable to many plant species. The present review chapter discusses the proposed molecular mechanisms of selective chromosome elimination in early embryogenesis and the effects of kinetochore component modifications on proper chromosome segregation. Finally, the advantages and limitations of the CENH3-mediated haploidization approach and its applications are highlighted.

Relative efficiency of chromosome elimination techniques for haploid induction parameters in triticale × wheat derived advanced generation through Zea mays- and Imperata cylindrica- mediated systems

The study was undertaken to evaluate the relative efficiency of doubled haploid production methods following the chromosome elimination approach utilizing maize- and Imperata cylindrica- mediated systems. For doubled haploid production, triticale x wheat derived recombinants in different generations comprised of F1, F2, F3, F4, BC1F1, BC1F2, BC1F3, BC1F4 and BC2F3 were used. The haploid embryos were cultured in the MS medium with different mineral constituents and hormones. All the generations responded for different haploid induction parameters following maize- and Imperata cylindrica- mediated systems. Advanced generations responded better than early generations for haploid induction through both systems. All the haploid induction parameters, Imperata cylindrical- were performed significantly better than maize.