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.

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.

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.

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.

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.

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.

Metabolism-based isolation of invasive glioblastoma cells with specific gene signatures and tumorigenic potential.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive brain tumor with rapid subclonal diversification, harboring molecular abnormalities that vary temporospatially, a contributor to therapy resistance. Fluorescence-guided neurosurgical resection utilizes the administration of 5-aminolevulinic acid (5-ALA) generating individually fluorescent tumor cells within a background population of non-neoplastic cells in the invasive tumor region. The aim of the study was to specifically isolate and interrogate the invasive GBM cell population using a novel 5-ALA-based method. METHODS: We have isolated the critical invasive GBM cell population by developing 5-ALA-based metabolic fluorescence-activated cell sorting. This allows purification and study of invasive cells from GBM without an overwhelming background "normal brain" signal to confound data. The population was studied using RNAseq, real-time PCR, and immunohistochemistry, with gene targets functionally interrogated on proliferation and migration assays using siRNA knockdown and known drug inhibitors. RESULTS: RNAseq analysis identifies specific genes such as SERPINE1 which is highly expressed in invasive GBM cells but at low levels in the surrounding normal brain parenchyma. siRNA knockdown and pharmacological inhibition with specific inhibitors of SERPINE1 reduced the capacity of GBM cells to invade in an in vitro assay. Rodent xenografts of 5-ALA-positive cells were established and serially transplanted, confirming tumorigenicity of the fluorescent patient-derived cells but not the 5-ALA-negative cells. CONCLUSIONS: Identification of unique molecular features in the invasive GBM population offers hope for developing more efficacious targeted therapies compared to targeting the tumor core and for isolating tumor subpopulations based upon intrinsic metabolic properties.

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.

Experimenting with Trinucleotide Repeats: Facts and Technical Issues.

Trinucleotide repeats are a peculiar class of microsatellites involved in many neurological as well as developmental disorders. Their propensity to generate very large expansions over time is supposedly due to their capacity to form specific secondary structures, such as imperfect hairpins, triple helices, or G-quadruplexes. These unusual structures were proposed to trigger expansions in vivo. Here, I review known technical issues linked to these structures, such as slippage during polymerase chain reaction and aberrant migration of long trinucleotide repeats during agarose gel electrophoresis. Our current understanding of interactions between trinucleotide repeat secondary structures and the mismatch-repair machinery is also quickly reviewed, and critical questions relevant to these interactions are addressed.

Quantifying Replication Fork Progression at CTG Repeats by 2D Gel Electrophoresis.

Physical separation of branched DNA from linear molecules is based on the difference of mobility of linear versus branched DNA during two-dimensional agarose gel electrophoresis. Structured DNA migrates as slower species when compared to linear DNA of similar molecular weight. Metabolic processes such as S phase replication or double strand-break repair may generate branched DNA molecules. Trinucleotide repeats are naturally prone to form secondary structures that can modify their migration through an agarose gel matrix. These structures may also interfere in vivo with replication, by slowing down replication-fork progression, transiently stalling forks, possibly leading to secondary structure such as Holliday junctions or hemicatenanes. Alternatively, reversed replication forks may occur following fork stalling, disrupting replication dynamics and modifying DNA migration on agarose gel. So although two-dimensional agarose gel electrophoresis theoretically allows to resolve a mixture of structured DNA molecules and quantify them by radioactive hybridization, its practical application to trinucleotide repeats faces some serious technical challenges.

Monitoring Double-Strand Break Repair of Trinucleotide Repeats Using a Yeast Fluorescent Reporter Assay.

Cells can repair a double-strand break (DSB) by homologous recombination if a homologous sequence is provided as a template. This can be achieved by classical gene conversion (with or without crossover) or by single-strand annealing (SSA) between two direct repeat sequences flanking the DSB. To initiate SSA, single-stranded regions are needed adjacent to the break, extending up to the direct repeats in such a way that complementary strands can anneal to each other to repair the DSB. In the present protocol, we describe a GFP reporter assay in Saccharomyces cerevisiae allowing for the quantification of nuclease efficacy at inducing a DSB, by monitoring the reconstitution of a functional GFP gene whose expression can be rapidly quantified by flow cytometry.

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.

Limitations of current in vitro models for testing the clinical potential of epigenetic inhibitors for treatment of pediatric ependymoma.

BACKGROUND: Epigenetic modifications have been shown to play an important role in the classification and pathogenesis of the pediatric brain tumor ependymoma, suggesting they are a potential therapeutic target. RESULTS: Agents targeting epigenetic modifications inhibited the growth and induced the death of ependymoma cells with variable efficiency. However, this was often not at clinically achievable doses. Additionally, DNA methylation profiling revealed a lack of similarity to primary ependymomas suggesting alterations were induced during culture. Toxicity to fetal neural stem cells was also seen at similar drug concentrations. CONCLUSIONS: Agents targeting epigenetic modifications were able to inhibit the growth and induced the death of ependymoma cells grown in vitro. However, many agents were only active at high doses, outside clinical ranges, and also resulted in toxicity to normal brain cells. The lack of similarity in DNA methylation profiles between cultured cells and primary ependymomas questions the validity of using in vitro cultured cells for pre-clinical analysis of agents targeting epigenetic mechanisms and suggests further investigation using models that are more appropriate should be undertaken before agents are taken forward for clinical testing. MATERIALS AND METHODS: The effects of agents targeting epigenetic modifications on the growth and death of a panel of ependymoma cell lines was investigated, as well as toxicity to normal fetal neural stem cells. The ependymoma cell lines were characterized using DNA methylation profiling.

TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats.

Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington's disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction.

A fast, sensitive and cost-effective method for nucleic acid detection using non-radioactive probes.

Nucleic acid detection and quantification using a labeled DNA probe is a very common molecular biology procedure. Here, we describe a new method, based on commonly used laboratory solutions, for nucleic acid hybridization and detection with digoxigenin-labeled DNA probes. The protocol described is faster, more sensitive and much cheaper than a standard protocol using commercial solutions. Comparison with a classical radioactive detection method shows that the latter exhibits less background and shows a greater linear response. Hence, the proposed protocol may be routinely performed for qualitative detection of nucleic acid, but when precise signal quantitation needs to be obtained, radioactive probe hybridization associated to phosphorimaging technology is more reliable.

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.