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SUMMARYAneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer and congenital syndromes. Here, we describe KaryoCreate (Karyotype CRISPR Engineered Aneuploidy Technology), a system that enables generation of chromosome-specific aneuploidies by co-expression of a sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to a mutant form of KNL1. We designed unique and highly specific sgRNAs for 19 out of 24 chromosomes. Expression of these sgRNAs with KNL1Mut-dCas9 leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny with an average efficiency of 8% and 12% for gains and losses, respectively (up to 20%), tested and validated across 9 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, a frequent occurrence in gastrointestinal cancers, promotes resistance to TGFβ, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe a novel technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.HighlightsWe designed sgRNAs targeting chromosome-specific centromeres across 19 human chromosomesKaryoCreate combines chromosome-specific centromeric sgRNAs with dCas9 fused to a mutant form of KNL1.KaryoCreate allows engineering gains and losses of specific human chromosomes.Engineered Chromosome 18q loss promotes tumor-associated phenotypes in colon-derived cells.KaryoCreate is a CRISPR-based technology to foster the study of centromere biology and aneuploidy.

Although the structure and function of the alphoid-tetO Human Artificial Chromosome (tetO-HAC) has been previously described in cell culture models and somatically in the mouse, in vivo persistence and stability throughout meiosis and across generations were not evaluated. Here we report germline transmission of a circular tetO-HAC across three mouse generations without observable health or reproductive deficiencies. Furthermore, we show that the tetO-HAC is maintained without selection as an episome and can be efficiently transmitted by both ova and sperm.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline forde novoDNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assembled the 101 kb humanHPRT1gene in yeast, delivered it to mouse embryonic stem cells, and showed expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells.Significance StatementMammalian genomes consist of a tiny proportion of relatively well-characterized coding regions and vast swaths of poorly characterized “dark matter” containing critical but much less well-defined regulatory sequences. Given the dominant role of noncoding DNA in common human diseases and traits, the interconnectivity of regulatory elements, and the importance of genomic context,de novodesign, assembly, and delivery can enable large-scale manipulation of these elements on a locus scale. Here we outline a pipeline forde novoassembly, delivery and expression of mammalian genes replete with native regulatory sequences. We expect this pipeline will be useful for dissecting the function of non-coding sequence variation in mammalian genomes.

Summary Telomere crisis occurs during tumorigenesis when depletion of the telomere reserve leads to frequent telomere fusions. The resulting dicentric chromosomes have been proposed to drive genome instability. Here, we examine the fate of dicentric human chromosomes in telomere crisis. We observed that dicentric chromosomes invariably persisted through mitosis and developed into 50–200 μm chromatin bridges connecting the daughter cells. Before their resolution at 3–20 hr after anaphase, the chromatin bridges induced nuclear envelope rupture in interphase, accumulated the cytoplasmic 3′ nuclease TREX1, and developed RPA-coated single stranded (ss) DNA. CRISPR knockouts showed that TREX1 contributed to the generation of the ssDNA and the resolution of the chromatin bridges. Post-crisis clones showed chromothripsis and kataegis, presumably resulting from DNA repair and APOBEC editing of the fragmented chromatin bridge DNA. We propose that chromothripsis in human cancer may arise through TREX1-mediated fragmentation of dicentric chromosomes formed in telomere crisis.

The mammalian chromosomes present specific sites of gaps or breaks, the common fragile sites (CFSs), when the cells are exposed to DNA replication stress or to some DNA binding compounds. CFSs span hundreds or thousands of kilobases. The analysis of these sequences has not definitively clarified the causes of their fragility. There is considerable evidence that CFSs are regions of late or slowed replication in the presence of sequence elements that have the propensity to form secondary structures, and that the cytogenetic expression of CFSs may be due to unreplicated DNA. In order to analyse the relationship between DNA replication time and fragility, in this work we have investigated the timing of replication of sequences mapping within two CFSs (FRA1H and FRA2G), of syntenic non-fragile sequences and of early and late replicating control sequences by using fluorescent in situ hybridization on interphase nuclei, conventional fluorescence microscopy and confocal microscopy. Our results indicate that the fragile sequences are slow replicating and that they enter G2 phase unreplicated with very high frequency. Thus these regions could sometimes reach mitosis unreplicated or undercondensed and be expressed as chromosome gaps/breakages.

Summary Modeling late-onset disorders such as Parkinson's disease (PD) using iPSC technology remains a challenge, as current differentiation protocols yield cells with the properties of fetal-stage cells. Here, we tested whether it is possible to accelerate aging in vitro to trigger late-onset disease phenotypes in an iPSC model of PD. In order to manipulate a factor that is involved in natural aging as well as in premature aging syndromes, we used telomere shortening as an age-inducing tool. We show that shortened telomeres result in age-associated as well as potentially disease-associated phenotypes in human pluripotent stem cell (hPSC)-derived midbrain dopamine (mDA) neurons. Our approach provides proof of concept for the further validation of telomere shortening as an induced-aging tool for late-onset-disease modeling.

Mouse telomeres have been suggested to resemble common fragile sites (CFS), showing disrupted TTAGGG fluorescent in situ hybridization signals after aphidicolin treatment. This “fragile” telomere phenotype is induced by deletion of TRF1, a shelterin protein that binds telomeric DNA and promotes efficient replication of the telomeric ds[TTAGGG]n tracts. Here we show that the chromosome-internal TTAGGG repeats present at human chromosome 2q14 form an aphidicolin-induced CFS. TRF1 binds to and stabilizes CFS 2q14 but does not affect other CFS, establishing 2q14 as the first CFS controlled by a sequence-specific DNA binding protein. The data show that telomeric DNA is inherently fragile regardless of its genomic position and imply that CFS can be caused by a specific DNA sequence.

KG-1 and its less differentiated subline KG-1a are leukemia cell lines used in research in a number of laboratories. The karyotypes of the two lines were initially identical. In the following years, further analysis revealed that the cell lines had acquired additional karyotypical abnormalities and differed in the presence of certain typical chromosomal rearrangements. To obtain cytogenetic authentication prior to the use of the two cell lines, we analyzed their karyotype by combining DAPI- and CMA-chromosome bandings and a fluorescence in situ hybridization (FISH)-based approach by using BAC clones useful for the identification of chromosome regions of interest. Sequences of the MYC, PLZF, RARA and BCR genes, that are known to play a critical role in leukemogenesis, and certain BAC clones mapped to five known common fragile sites (CFS) were used for the FISH analysis. A telomeric probe (TTAGGG)n and a set of BAC clones were used to characterize the marker chromosome der(1) that was observed in the cell line KG-1a. The existence of notable differences between the karyotype of the KG-1a cell line previously described, and that described in this study, demonstrate that the use of established cancer cell lines should be preceded by cytogenetic and/or molecular characterization.

Genome amplification is often observed in human tumors. The breakage-fusion-bridge (BFB) cycle is the mechanism that often underlies duplicated regions. Some research has indicated common fragile sites (CFS) as possible sites of chromosome breakages at the origin of BFB cycles. Here we searched two human genome regions known as amplification hot spots for any DNA copy number amplifications by analyzing 21 cancer cell lines to investigate the relationship between genomic fragility and amplification. We identified a duplicated region on a chromosomes der(2) present in the karyotype of two analysed leukemia cell lines K562. The two duplicated regions are organized into large palindromes, which suggests that one BFB cycle has occurred. Our findings show that the three breakpoints are localized in the sequence of three CFSs: FRA2H (2q32.1-q32.2), which here has been characterized molecularly; FRA2S (2q22.3-q23.3), a newly localized aphidicolin inducible CFS; and FRA2G (2q24.3-q31).

Common fragile sites (CFS) are specific regions of the mammalian chromosomes that are particularly prone to gaps and breaks. They are a cause of genome instability, and the location of many CFS correlates with breakpoints of aberrations recurrent in some cancers. The molecular characterization of some CFS has not clarified the causes of their fragility. In this work, by using fluorescence in situ hybridization analysis with BAC and PAC clones, we determined the DNA sequence of the CFS FRA7B. The FRA7B sequence was then analyzed to identify coding sequences and some structural features possibly involved in fragility. FRA7B spans about 12.2 megabases, and is therefore one of the largest CFS analyzed. It maps at the 7p21.3-22.3 chromosome bands, therefore at the interface of G- and R-band regions that are probably difficult to replicate. A 90-kilobase long sequence that presents very high flexibility values was identified at the very beginning of the more fragile CFS region. Three large genes (THSD7A, SDK1, and MAD1L1) and two miRNA genes (MIRN589 and MIRN339) map in the fragile region. The chromosome band 7p22 is a recurrent breakpoint in chromosome abnormalities in different types of neoplasm. FRA7B is the first characterized CFS located in a chromosome terminal region.

Common fragile sites (CFSs) are chromosome regions that exhibit gaps and breaks when the cells are exposed to replication stress and to some DNA-binding compounds. In cancer cells, the CFSs are frequently involved in recurrent chromosome rearrangements. Furthermore, altered expression of associated genes, known or potential oncogenes, and tumor-suppressor genes has often been observed. Seventeen of the 88 listed CFSs have been analyzed at the molecular level, but the basis of their fragility has not been clarified. In the present work, the nine genes TGFB2, IARS2, MARK1, TAF1A, TP53BP2, ADPRT, including a very large gene ESRRG and two microRNA genes, MIRN194-1 and MIRN215, localized in the fragile site FRA1H, were investigated by polymerase chain reaction (PCR) for homozygous deletions and by real-time PCR for modification or loss of gene expression in a panel of 19 cancer cell lines. The expression level of five (ESRRG, TGFB2, MIRN194-1, MIRN215, and MARK1) of the nine genes studied presented significant modifications in some of the 19 examined tumor-derived cell lines compared to their normal control tissues. Because of their function, these genes could have a role in neoplastic transformation.