Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations.

Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR-Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)33. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions.

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.

Time-resolved microfluidics unravels individual cellular fates during double-strand break repair.

BACKGROUND: Double-strand break repair (DSBR) is a highly regulated process involving dozens of proteins acting in a defined order to repair a DNA lesion that is fatal for any living cell. Model organisms such as Saccharomyces cerevisiae have been used to study the mechanisms underlying DSBR, including factors influencing its efficiency such as the presence of distinct combinations of microsatellites and endonucleases, mainly by bulk analysis of millions of cells undergoing repair of a broken chromosome. Here, we use a microfluidic device to demonstrate in yeast that DSBR may be studied at a single-cell level in a time-resolved manner, on a large number of independent lineages undergoing repair. RESULTS: We used engineered S. cerevisiae cells in which GFP is expressed following the successful repair of a DSB induced by Cas9 or Cpf1 endonucleases, and different genetic backgrounds were screened to detect key events leading to the DSBR efficiency. Per condition, the progenies of 80-150 individual cells were analyzed over 24 h. The observed DSBR dynamics, which revealed heterogeneity of individual cell fates and their contributions to global repair efficacy, was confronted with a coupled differential equation model to obtain repair process rates. Good agreement was found between the mathematical model and experimental results at different scales, and quantitative comparisons of the different experimental conditions with image analysis of cell shape enabled the identification of three types of DSB repair events previously not recognized: high-efficacy error-free, low-efficacy error-free, and low-efficacy error-prone repair. CONCLUSIONS: Our analysis paves the way to a significant advance in understanding the complex molecular mechanism of DSB repair, with potential implications beyond yeast cell biology. This multiscale and multidisciplinary approach more generally allows unique insights into the relation between in vivo microscopic processes within each cell and their impact on the population dynamics, which were inaccessible by previous approaches using molecular genetics tools alone.

Trinucleotide repeat instability during double-strand break repair: from mechanisms to gene therapy.

Trinucleotide repeats are a particular class of microsatellites whose large expansions are responsible for at least two dozen human neurological and developmental disorders. Slippage of the two complementary DNA strands during replication, homologous recombination or DNA repair is generally accepted as a mechanism leading to repeat length changes, creating expansions and contractions of the repeat tract. The present review focuses on recent developments on double-strand break repair involving trinucleotide repeat tracts. Experimental evidences in model organisms show that gene conversion and break-induced replication may lead to large repeat tract expansions, while frequent contractions occur either by single-strand annealing between repeat ends or by gene conversion, triggering near-complete contraction of the repeat tract. In the second part of this review, different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared. Relative efficacies and specificities of these nucleases will be discussed, as well as their potential strengths and weaknesses for possible future gene therapy of these dramatic disorders.

Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.

Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.

Shortening trinucleotide repeats using highly specific endonucleases: a possible approach to gene therapy?

Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.

Genome-wide replication landscape of Candida glabrata.

BACKGROUND: The opportunistic pathogen Candida glabrata is a member of the Saccharomycetaceae yeasts. Like its close relative Saccharomyces cerevisiae, it underwent a whole-genome duplication followed by an extensive loss of genes. Its genome contains a large number of very long tandem repeats, called megasatellites. In order to determine the whole replication program of the C. glabrata genome and its general chromosomal organization, we used deep-sequencing and chromosome conformation capture experiments. RESULTS: We identified 253 replication fork origins, genome wide. Centromeres, HML and HMR loci, and most histone genes are replicated early, whereas natural chromosomal breakpoints are located in late-replicating regions. In addition, 275 autonomously replicating sequences (ARS) were identified during ARS-capture experiments, and their relative fitness was determined during growth competition. Analysis of ARSs allowed us to identify a 17-bp consensus, similar to the S. cerevisiae ARS consensus sequence but slightly more constrained. Megasatellites are not in close proximity to replication origins or termini. Using chromosome conformation capture, we also show that early origins tend to cluster whereas non-subtelomeric megasatellites do not cluster in the yeast nucleus. CONCLUSIONS: Despite a shorter cell cycle, the C. glabrata replication program shares unexpected striking similarities to S. cerevisiae, in spite of their large evolutionary distance and the presence of highly repetitive large tandem repeats in C. glabrata. No correlation could be found between the replication program and megasatellites, suggesting that their formation and propagation might not be directly caused by replication fork initiation or termination.

Dynamics of a combined Medea-underdominant population transformation system.

BACKGROUND: Transgenic constructs intended to be stably established at high frequencies in wild populations have been demonstrated to "drive" from low frequencies in experimental insect populations. Linking such population transformation constructs to genes which render them unable to transmit pathogens could eventually be used to stop the spread of vector-borne diseases like malaria and dengue. RESULTS: Generally, population transformation constructs with only a single transgenic drive mechanism have been envisioned. Using a theoretical modelling approach we describe the predicted properties of a construct combining autosomal Medea and underdominant population transformation systems. We show that when combined they can exhibit synergistic properties which in broad circumstances surpass those of the single systems. CONCLUSION: With combined systems, intentional population transformation and its reversal can be achieved readily. Combined constructs also enhance the capacity to geographically restrict transgenic constructs to targeted populations. It is anticipated that these properties are likely to be of particular value in attracting regulatory approval and public acceptance of this novel technology.

Purification of G1 daughter cells from different Saccharomycetes species through an optimized centrifugal elutriation procedure.

Centrifugal elutriation discriminates cells according to their sedimentation coefficients, generating homogeneous samples well suited for genomic comparative approaches. It can, for instance, isolate G1 daughter cells from a Saccharomyces cerevisiae unsynchronized population, alleviating ageing and cell-cycle biases when conducting genome-wide/single-cell studies. The present report describes a straightforward and robust procedure to determine whether a cell population of virtually any yeast species can be efficiently elutriated, while offering solutions to optimize success. This approach was used to characterize elutriation parameters and S-phase progression of four yeast species (S. cerevisiae, Candida glabrata, Lachancea kluyveri and Pichia sorbitophila) and could theoretically be applied to any culture of single, individual cells.

Detection and characterization of megasatellites in orthologous and nonorthologous genes of 21 fungal genomes.

Megasatellites are large DNA tandem repeats, originally described in Candida glabrata, in protein-coding genes. Most of the genes in which megasatellites are found are of unknown function. In this work, we extended the search for megasatellites to 20 additional completely sequenced fungal genomes and extracted 216 megasatellites in 203 out of 142,121 genes, corresponding to the most exhaustive description of such genetic elements available today. We show that half of the megasatellites detected encode threonine-rich peptides predicted to be intrinsically disordered, suggesting that they may interact with several partners or serve as flexible linkers. Megasatellite motifs were clustered into several families. Their distribution in fungal genes shows that different motifs are found in orthologous genes and similar motifs are found in unrelated genes, suggesting that megasatellite formation or spreading does not necessarily track the evolution of their host genes. Altogether, these results suggest that megasatellites are created and lost during evolution of fungal genomes, probably sharing similar functions, although their primary sequences are not necessarily conserved.

Can genome editing help transitioning to agroecology?

Meeting the challenges of agroecological transition in a context of climate change requires the use of various strategies such as biological regulations, adapted animal and plant genotypes, diversified production systems, and digital technologies. Seeds and plants, through plant breeding, play a crucial role in driving these changes. The emergence of genome editing presents a new opportunity in plant breeding practices. However, like any technological revolution involving living organisms, it is essential to assess its potential contributions, limits, risks, socio-economic implications, and the associated controversies. This article aims to provide a comprehensive review of scientific knowledge on genome editing for agroecological transition, drawing on multidisciplinary approaches encompassing biological, agronomic, economic, and social sciences.

Dynamic evolution of megasatellites in yeasts.

Megasatellites are a new family of long tandem repeats, recently discovered in the yeast Candida glabrata. Compared to shorter tandem repeats, such as minisatellites, megasatellite motifs range in size from 135 to more than 300 bp, and allow calculation of evolutionary distances between individual motifs. Using divergence based on nucleotide substitutions among similar motifs, we determined the smallest distance between two motifs, allowing their subsequent clustering. Motifs belonging to the same cluster are recurrently found in different megasatellites located on different chromosomes, showing transfer of genetic information between megasatellites. In comparison, evolution of the few similar tandem repeats in Saccharomyces cerevisiae FLO genes mainly involves subtelomeric homologous recombination. We estimated selective constraints acting on megasatellite motifs and their host genes, and found that motifs are under strong purifying selection. Surprisingly, motifs inserted within pseudogenes are also under purifying selection, whereas the pseudogenes themselves evolve neutrally. We propose that megasatellite motifs propagate by a combination of three different molecular mechanisms: (i) gene duplication, (ii) ectopic homologous recombination and (iii) transfer of motifs from one megasatellite to another one. These mechanisms actively cooperate to create new megasatellites, that may play an important role in the adaptation of Candida glabrata to its human host.

Megasatellite formation and evolution in vertebrate genes.

Since formation of the first proto-eukaryotes, gene repertoire and genome complexity have significantly increased. Among genetic elements responsible for this increase are tandem repeats. Here we describe a genome-wide analysis of large tandem repeats, called megasatellites, in 58 vertebrate genomes. Two bursts occurred, one after the radiation between Agnatha and Gnathostomata fishes and the second one in therian mammals. Megasatellites are enriched in subtelomeric regions and frequently encoded in genes involved in transcription regulation, intracellular trafficking, and cell membrane metabolism, reminiscent of what is observed in fungus genomes. The presence of many introns within young megasatellites suggests that an exon-intron DNA segment is first duplicated and amplified before accumulation of mutations in intronic parts partially erases the megasatellite in such a way that it becomes detectable only in exons. Our results suggest that megasatellite formation and evolution is a dynamic and still ongoing process in vertebrate genomes.

Functional variability in adhesion and flocculation of yeast megasatellite genes.

Megasatellites are large tandem repeats found in all fungal genomes but especially abundant in the opportunistic pathogen Candida glabrata. They are encoded in genes involved in cell-cell interactions, either between yeasts or between yeast and human cells. In the present work, we have been using an iterative genetic system to delete several Candida glabrata megasatellite-containing genes and found that 2 of them were positively involved in adhesion to epithelial cells, whereas 3 genes negatively controlled adhesion. Two of the latter, CAGL0B05061g or CAGL0A04851g, were also negative regulators of yeast-to-yeast adhesion, making them central players in controlling Candida glabrata adherence properties. Using a series of synthetic Saccharomyces cerevisiae strains in which the FLO1 megasatellite was replaced by other tandem repeats of similar length but different sequences, we showed that the capacity of a strain to flocculate in liquid culture was unrelated to its capacity to adhere to epithelial cells or to invade agar. Finally, to understand how megasatellites were initially created and subsequently expanded, an experimental evolution system was set up, in which modified yeast strains containing different megasatellite seeds were grown in bioreactors for more than 200 generations and selected for their ability to sediment at the bottom of the culture tube. Several flocculation-positive mutants were isolated. Functionally relevant mutations included general transcription factors as well as a 230-kbp segmental duplication.

Mathematical models for the study of HIV spread and control amongst men who have sex with men.

For a quarter of century, mathematical models have been used to study the spread and control of HIV amongst men who have sex with men (MSM). We searched MEDLINE and EMBASE databases up to the end of 2010 and reviewed this literature to summarise the methodologies used, key model developments, and the recommended strategies for HIV control amongst MSM. Of 742 studies identified, 127 studies met the inclusion criteria. Most studies employed deterministic modelling methods (80%). Over time we saw an increase in model complexity regarding antiretroviral therapy (ART), and a corresponding decrease in complexity regarding sexual behaviours. Formal estimation of model parameters was carried out in only a small proportion of the studies (22%) while model validation was considered by an even smaller proportion (17%), somewhat reducing confidence in the findings from the studies. Nonetheless, a number of common conclusions emerged, including (1) identification of the importance of assumptions regarding changes in infectivity and sexual contact rates on the impact of ART on HIV incidence, that subsequently led to empirical studies to gather these data, and (2) recommendation that multiple strategies would be required for effective HIV control amongst MSM. The role of mathematical models in studying epidemics is clear, and the lack of formal inference and validation highlights the need for further developments in this area. Improved methodologies for parameter estimation and systematic sensitivity analysis will help generate predictions that more fully express uncertainty, allowing better informed decision making in public health.

Megasatellites: a new class of large tandem repeats discovered in the pathogenic yeast Candida glabrata.

Megasatellites are DNA tandem arrays made of large motifs; they were discovered in the yeast Candida glabrata. They are widespread in this species (40 copies) but are not found in any other hemiascomycete so far, raising the intriguing question of their origin. They are found mainly in genes encoding cell wall products, suggesting that megasatellites were selected for a function linked to cell-cell adhesion or to pathogenicity. Their putative role in promoting genome rearrangements by interfering with DNA replication will also be discussed.

The Startling Role of Mismatch Repair in Trinucleotide Repeat Expansions.

Trinucleotide repeats are a peculiar class of microsatellites whose expansions are responsible for approximately 30 human neurological or developmental disorders. The molecular mechanisms responsible for these expansions in humans are not totally understood, but experiments in model systems such as yeast, transgenic mice, and human cells have brought evidence that the mismatch repair machinery is involved in generating these expansions. The present review summarizes, in the first part, the role of mismatch repair in detecting and fixing the DNA strand slippage occurring during microsatellite replication. In the second part, key molecular differences between normal microsatellites and those that show a bias toward expansions are extensively presented. The effect of mismatch repair mutants on microsatellite expansions is detailed in model systems, and in vitro experiments on mismatched DNA substrates are described. Finally, a model presenting the possible roles of the mismatch repair machinery in microsatellite expansions is proposed.

Alternative DNA Structures In Vivo: Molecular Evidence and Remaining Questions.

Duplex DNA naturally folds into a right-handed double helix in physiological conditions. Some sequences of unusual base composition may nevertheless form alternative structures, as was shown for many repeated sequences in vitro However, evidence for the formation of noncanonical structures in living cells is difficult to gather. It mainly relies on genetic assays demonstrating their function in vivo or through genetic instability reflecting particular properties of such structures. Efforts were made to reveal their existence directly in a living cell, mainly by generating antibodies specific to secondary structures or using chemical ligands selected for their affinity to these structures. Among secondary structure-forming DNAs are G-quadruplexes, human fragile sites containing minisatellites, AT-rich regions, inverted repeats able to form cruciform structures, hairpin-forming CAG/CTG triplet repeats, and triple helices formed by homopurine-homopyrimidine GAA/TTC trinucleotide repeats. Many of these alternative structures are involved in human pathologies, such as neurological or developmental disorders, as in the case of trinucleotide repeats, or cancers triggered by translocations linked to fragile sites. This review will discuss and highlight evidence supporting the formation of alternative DNA structures in vivo and will emphasize the role of the mismatch repair machinery in binding mispaired DNA duplexes, triggering genetic instability.

Effect of cropping systems and climate on soil physical characteristics, field crop emergence and yield: A dataset from a 19-year field experiment.

A long-term field experiment was conducted from 1989 to 2007 in northern France in a loamy soil to assess the cumulative effects of cropping systems (CSs) on soil compaction, soil porosity, soil structure, crop emergence and yield. Three CSs, including different crop rotations and cultivations (early or late sowing and harvesting), were compared. CS I was the succession of spring pea/winter wheat/oilseed rape (flax from 2001)/winter wheat while CSs II and III were the succession of sugar beet/winter wheat/maize/winter wheat. The latter two CSs consisted of different sowing dates, based on two distinct decision rules aimed at minimizing the risk of soil compaction in the CS II or maximizing the duration of the crop in the CS III. The tillage system was only mouldboard ploughing up to 2000 while a new treatment with superficial tillage (i.e. at 6 cm depth) was integrated since then into the experiment to compare the effects of annual ploughing and reduced tillage on changes in soil structure over time. Soil water content was measured for each field operation by taking samples every 0.05 m up to a depth of 0.30 m in the topsoil. Soil compaction and soil structure was evaluated after each sowing using a morphological approach and soil bulk density measurements. The ''profil cultural'' method was used to map soil structure variations in the topsoil below the seedbed. Dry bulk density was measured with a gamma-ray transmission probe. Seedling emergence rates and crop yield were also measured in relation to CSs. This dataset represents an important description of the changes in the soil compaction level, crop emergence rates and yield, in relation to CSs and climate, and the overall impact on seedbed structure variations for major field crops under northern France conditions. This information can be used as input variables of several soil-crop models aiming at evaluating the impact of CSs and climate on soil compaction and seedbed structures.

Genome evolution in yeasts.

Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.