Increased RPA1 gene dosage affects genomic stability potentially contributing to 17p13.3 duplication syndrome

A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage response (DDR). Here, we show that cell lines from patients with duplications specifically incorporating RPA1 exhibit a different although characteristic spectrum of DDR defects including abnormal S phase distribution, attenuated DNA double strand break (DSB)-induced RAD51 chromatin retention, elevated genomic instability, and increased sensitivity to DNA damaging agents. Using controlled conditional over-expression of RPA1 in a human model cell system, we also see attenuated DSB-induced RAD51 chromatin retention. Furthermore, we find that transient over-expression of RPA1 can impact on homologous recombination (HR) pathways following DSB formation, favouring engagement in aberrant forms of recombination and repair. Our data identifies unanticipated defects in the DDR associated with duplications in 17p13.3 in humans involving modest RPA1 over-expression

I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species

EMSY overexpression disrupts the BRCA2/RAD51 pathway in the DNA-damage response: implications for chromosomal instability/recombination syndromes as checkpoint diseases

EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer. It encodes a nuclear protein that binds to the BRCA2 N-terminal domain implicated in chromatin/transcription regulation, but when sporadically amplified/overexpressed, increased EMSY level represses BRCA2 transactivation potential and induces chromosomal instability, mimicking the activity of BRCA2 mutations in the development of hereditary breast/ovarian cancer. In addition to chromatin/transcription regulation, EMSY may also play a role in the DNA-damage response, suggested by its ability to localize at chromatin sites of DNA damage/repair. This implies that EMSY overexpression may also repress BRCA2 in DNA-damage replication/checkpoint and recombination/repair, coordinated processes that also require its interacting proteins: PALB2, the partner and localizer of BRCA2; RPA, replication/checkpoint protein A; and RAD51, the inseparable recombination/repair enzyme. Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression. Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA. We extensively discuss the molecular details of how this can happen to ascertain its implications for a novel recombination mechanism apparently conceived as checkpoint rather than a DNA repair system for cell division, survival, death, and human diseases, including the tissue specificity of cancer predisposition, which may renew our thinking about targeted therapy and prevention

LTR Retrotransposons in Fungi

Transposable elements with long terminal direct repeats (LTR TEs) are one of the best studied groups of mobile elements. They are ubiquitous elements present in almost all eukaryotic genomes. Their number and state of conservation can be a highlight of genome dynamics. We searched all published fungal genomes for LTR-containing retrotransposons, including both complete, functional elements and remnant copies. We identified a total of over 66,000 elements, all of which belong to the Ty1/Copia or Ty3/Gypsy superfamilies. Most of the detected Gypsy elements represent Chromoviridae, i.e. they carry a chromodomain in the pol ORF. We analyzed our data from a genome-ecology perspective, looking at the abundance of various types of LTR TEs in individual genomes and at the highest-copy element from each genome. The TE content is very variable among the analyzed genomes. Some genomes are very scarce in LTR TEs (<50 elements), others demonstrate huge expansions (>8000 elements). The data shows that transposon expansions in fungi usually involve an increase both in the copy number of individual elements and in the number of element types. The majority of the highest-copy TEs from all genomes are Ty3/Gypsy transposons. Phylogenetic analysis of these elements suggests that TE expansions have appeared independently of each other, in distant genomes and at different taxonomical levels. We also analyzed the evolutionary relationships between protein domains encoded by the transposon pol ORF and we found that the protease is the fastest evolving domain whereas reverse transcriptase and RNase H evolve much slower and in correlation with each other

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology

Targeted plant improvement through genome editing: from laboratory to field

This review illustrates how far we have come since the emergence of GE technologies and how they could be applied to obtain superior and sustainable crop production. The main challenges of today's agriculture are maintaining and raising productivity, reducing its negative impact on the environment, and adapting to climate change. Efficient plant breeding can generate elite varieties that will rapidly replace obsolete ones and address ongoing challenges in an efficient and sustainable manner. Site-specific genome editing in plants is a rapidly evolving field with tangible results. The technology is equipped with a powerful toolbox of molecular scissors to cut DNA at a pre-determined site with different efficiencies for designing an approach that best suits the objectives of each plant breeding strategy. Genome editing (GE) not only revolutionizes plant biology, but provides the means to solve challenges related to plant architecture, food security, nutrient content, adaptation to the environment, resistance to diseases and production of plant-based materials. This review illustrates how far we have come since the emergence of these technologies and how these technologies could be applied to obtain superior, safe and sustainable crop production. Synergies of genome editing with other technological platforms that are gaining significance in plants lead to an exciting new, post-genomic era for plant research and production. In previous months, we have seen what global changes might arise from one new virus, reminding us of what drastic effects such events could have on food production. This demonstrates how important science, technology, and tools are to meet the current time and the future. Plant GE can make a real difference to future sustainable food production to the benefit of both mankind and our environment.European Cooperation in Science and Technology (COST) CA18111info:eu-repo/semantics/publishedVersio