190 research outputs found
L'enginyeria genĂČmica, l'Ășltima revoluciĂł en la millora genĂštica de les plantes cultivades
La millora genĂštica de les plantes cultivades ha permĂšs a les societats humanes obtenir suficient quantitat d'aliments de qualitat al llarg de la histĂČria. Aquest procĂ©s, que comença al neolĂtic, s'ha anat tecnificant i fent mĂ©s eficient a mesura que la ciĂšncia avançava. La mutagĂšnesi a mitjan segle XX i la transgĂšnia al final dels anys noranta del mateix segle, entre altres tĂšcniques, han permĂšs fer un salt endavant en la millora genĂštica. Recentment s'han posat a punt tĂšcniques de mutagĂšnesi dirigida amb nucleases especĂfiques que poden revolucionar la millora genĂštica. En particular, la mutagĂšnesi amb el sistema CRISPR/Cas9 estĂ permetent ja obtenir noves variants al·lĂšliques amb una eficiĂšncia i precisiĂł sense precedents. Malgrat l'interĂšs evident d'aquestes tĂšcniques, el seu Ăšxit dependrĂ , en gran manera, de la regulaciĂł que s'apliqui a les plantes obtingudes, i en particular de si la legislaciĂł europea les considera transgĂšniques o no. En aquest article analitzem l'interĂšs d'aquestes tĂšcniques a la llum de la histĂČria de la millora genĂštica de les plantes i en discutim la possible regulaciĂł.La mejora genĂ©tica de las plantas cultivadas ha permitido a las sociedades humanas obtener suficientes alimentos de calidad a lo largo de la historia. Este proceso, que empieza en el NeolĂtico, ha ido tecnificĂĄndose y haciĂ©ndose mĂĄs eficiente en paralelo al avance cientĂfico. La mutagĂ©nesis desde mediados del siglo XX y la transgenia desde finales de los años noventa del mismo siglo, entre otras tĂ©cnicas, permitieron dar un salto cualitativo en la mejora genĂ©tica. Recientemente se han puesto a punto tĂ©cnicas de mutagĂ©nesis dirigida con nucleasas especĂficas que pueden revolucionar la mejora genĂ©tica. En particular, la mutagĂ©nesis usando el sistema CRISPR/Cas9 estĂĄ permitiendo ya obtener nuevas variantes alĂ©licas con una eficiencia y precisiĂłn sin precedentes. Aunque el interĂ©s en estas tĂ©cnicas es evidente, su Ă©xito dependerĂĄ en gran medida de la regulaciĂłn que se aplique a los productos obtenidos de estas plantas, y en particular de si la legislaciĂłn europea las considera transgĂ©nicas o no. En este artĂculo analizamos el interĂ©s de estas tĂ©cnicas a la luz de la historia de la mejora genĂ©tica de las plantas y discutimos su posible regulaciĂłn.Plant breeding has allowed human societies to secure the production of food of good quality throughout history. This process, which started in the Neolithic, has become increasingly technologically based and efficient in step with the advance of scientific knowledge. Mutagenesis, since the mid 20th century, and transgenic plants since the late 1990s, among other techniques, allowed a qualitative leap forward in plant breeding. Recently, new site-directed mutagenesis techniques have been developed which may have a large impact on plant breeding. In particular, CRISPR/Cas9 mutagenesis
approaches are already allowing new alleles to be obtained with unprecedented efficiency and precision. In spite of the obvious interest of these techniques, their success in plant breeding will greatly depend on the regulation applied to the plants which are obtained and more specifically on whether or not these plants will be considered GMOs. In this article we describe the interest of these new techniques and discuss their possible regulation
MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens
MSH2 is a central component of the mismatch repair pathway that targets mismatches arising during DNA replication, homologous recombination (HR) and in response to genotoxic stresses. Here, we describe the function of MSH2 in the moss Physcomitrella patens, as deciphered by the analysis of loss of function mutants. Ppmsh2 mutants display pleiotropic growth and developmental defects, which reflect genomic instability. Based on loss of function of the APT gene, we estimated this mutator phenotype to be at least 130 times higher in the mutants than in wild type. We also found that MSH2 is involved in some but not all the moss responses to genotoxic stresses we tested. Indeed, the Ppmsh2 mutants were more tolerant to cisplatin and show higher sensitivity to UV-B radiations. PpMSH2 gene involvement in HR was studied by assessing gene targeting (GT) efficiency with homologous and homeologous sequences. GT efficiency with homologous sequences was slightly decreased in the Ppmsh2 mutant compared with wild type. Strikingly GT efficiency with homeologous sequences decreased proportionally to sequence divergence in the wild type whereas it remained unaffected in the mutants. Those results demonstrate the role of PpMSH2 in the maintenance of genome integrity and in homologous and homeologous recombinatio
Genome engineering and plant breeding : impact on trait discovery and development
Key message: New tools for the precise modification of crops genes are now available for the engineering of new ideotypes. A future challenge in this emerging field of genome engineering is to develop efficient methods for allele mining. Abstract: Genome engineering tools are now available in plants, including major crops, to modify in a predictable manner a given gene. These new techniques have a tremendous potential for a spectacular acceleration of the plant breeding process. Here, we discuss how genetic diversity has always been the raw material for breeders and how they have always taken advantage of the best available science to use, and when possible, increase, this genetic diversity. We will present why the advent of these new techniques gives to the breeders extremely powerful tools for crop breeding, but also why this will require the breeders and researchers to characterize the genes underlying this genetic diversity more precisely. Tackling these challenges should permit the engineering of optimized alleles assortments in an unprecedented and controlled way
MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens
The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and developmen
A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens
CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing
The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in Physcomitrella patens
The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3âČ non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells
Risk assessment of new sequencing data on GM maize event MIR604
In 2009 and 2010, the EFSA GMO Panel concluded the assessment of genetically modified (GM) maizes MIR604, MIR604 Ă GA21, MIR604 Ă Bt11 and MIR604 Ă GA21 Ă Bt11. These maizes were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 23 July 2015, the European Commission (EC) received from Syngenta new nucleic acid sequencing data on maize event MIR604 and updated bioinformatic analyses using the new sequencing data. EC tasked EFSA to analyse these data and to indicate whether the previous conclusions of the EFSA GMO Panel on the above-listed GM maizes remain valid. The EFSA GMO Panel used the appropriate principles described in its guidelines for the risk assessment of GM plants to analyse the received data. The new sequencing data indicated a single base pair difference compared to the sequencing data originally provided, located in a non-coding region of the insert. which had already been present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment remain valid. The new sequencing data and the bioinformatic analyses performed on the new sequence did not give rise to safety issues. Therefore, the GMO Panel concludes that the original risk assessment of event MIR604 as a single and as a part of stacked events remains valid
Assessment of genetically modified oilseed rape GT73 for renewal authorisation under Regulation (EC) No 1829/2003 (application EFSAâGMOâRXâ002)
Following the submission of application EFSA-GMO-RX-002 under Regulation (EC) No 1829/2003 from Monsanto Company, the Panel on Genetically Modified Organisms of EFSA (GMO) was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the herbicide-tolerant genetically modified oilseed rape GT73. The data received in the context of this renewal application contained post-market environmental monitoring reports, a systematic search and evaluation of literature, updated bioinformatic analyses and additional documents or studies performed by or on behalf of the applicant. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Under the assumption that the DNA sequence of the event in oilseed rape GT73 considered for renewal of authorisation is identical to the sequence of the originally assessed event, the GMO Panel concludes that there is no evidence in renewal application EFSA-GMO-RX-002 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on oilseed rape GT73
Applicability of the EFSA Opinion on site-directed nucleases type 3 for the safety assessment of plants developed using site-directed nucleases type 1 and 2 and oligonucleotide directed mutagenesis
© 2020 European Food Safety Authority.The European Commission requested the EFSA Panel on Genetically Modified Organisms (GMO) to assess whether section 4 (hazard identification) and the conclusions of EFSA's Scientific opinion on the risk assessment of plants developed using zinc finger nuclease type 3 technique (ZFNâ3) and other siteâdirected nucleases (SDN) with similar function are valid for plants developed via SDNâ1, SDNâ2 and oligonucleotideâdirected mutagenesis (ODM). In delivering this Opinion, the GMO Panel compared the hazards associated with plants produced via SDNâ1, SDNâ2 and ODM with those associated with plants obtained via both SDNâ3 and conventional breeding. Unlike for SDNâ3 methods, the application of SDNâ1, SDNâ2 and ODM approaches aims to modify genomic sequences in a way which can result in plants not containing any transgene, intragene or cisgene. Consequently, the GMO Panel concludes that those considerations which are specifically related to the presence of a transgene, intragene or cisgene included in section 4 and the conclusions of the Opinion on SDNâ3 are not relevant to plants obtained via SDNâ1, SDNâ2 or ODM as defined in this Opinion. Overall, the GMO Panel did not identify new hazards specifically linked to the genomic modification produced via SDNâ1, SDNâ2 or ODM as compared with both SDNâ3 and conventional breeding. Furthermore, the GMO Panel considers that the existing Guidance for risk assessment of food and feed from genetically modified plants and the Guidance on the environmental risk assessment of genetically modified plants are sufficient but are only partially applicable to plants generated via SDNâ1, SDNâ2 or ODM. Indeed, those guidance documentsâ requirements that are linked to the presence of exogenous DNA are not relevant for the risk assessment of plants developed via SDNâ1, SDNâ2 or ODM approaches if the genome of the final product does not contain exogenous DNA
- âŠ