Beyond seek and destroy: How to generate allelic series using genome editing tools

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations

Manipulation of Meiotic Recombination to Hasten Crop Improvement

International audienceSimple Summary Harnessing the natural and induced diversity existing in plant genetic resources is fundamental for building future crops more sober in fertilizers, water, and pesticides that can cope with climate instability while yielding healthier and more nutritious products. The essence of plant breeding is to combine favorable traits in crossing parental varieties to select novel performing associations amongst the progenies. These associations are the product of recombination between the parental chromosomes occurring during meiosis, mainly by a reciprocal DNA exchange called Cross Over (CO). However, recombination does not occur randomly along the chromosomes, and COs are limited in number often hampering the desired associations of favorable traits. This review surveys the recent advances in methods for achieving a stimulation and/or a redistribution of meiotic COs along the parental chromosomes and targeting COs specifically at desired chromosomal sites. Reciprocal (cross-overs = COs) and non-reciprocal (gene conversion) DNA exchanges between the parental chromosomes (the homologs) during meiotic recombination are, together with mutation, the drivers for the evolution and adaptation of species. In plant breeding, recombination combines alleles from genetically diverse accessions to generate new haplotypes on which selection can act. In recent years, a spectacular progress has been accomplished in the understanding of the mechanisms underlying meiotic recombination in both model and crop plants as well as in the modulation of meiotic recombination using different strategies. The latter includes the stimulation and redistribution of COs by either modifying environmental conditions (e.g., T degrees), harnessing particular genomic situations (e.g., triploidy in Brassicaceae), or inactivating/over-expressing meiotic genes, notably some involved in the DNA double-strand break (DSB) repair pathways. These tools could be particularly useful for shuffling diversity in pre-breeding generations. Furthermore, thanks to the site-specific properties of genome editing technologies the targeting of meiotic recombination at specific chromosomal regions nowadays appears an attainable goal. Directing COs at desired chromosomal positions would allow breaking linkage situations existing between favorable and unfavorable alleles, the so-called linkage drag, and accelerate genetic gain. This review surveys the recent achievements in the manipulation of meiotic recombination in plants that could be integrated into breeding schemes to meet the challenges of deploying crops that are more resilient to climate instability, resistant to pathogens and pests, and sparing in their input requirements

Unravelling nutrient exchange in ectomycorrhizal symbiosis

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CRISPR/Cas9-Targeted Knockout of Rice Susceptibility Genes OsDjA2 and OsERF104 Reveals Alternative Sources of Resistance to Pyricularia oryzae

International audienceRice genes OsDjA2 and OsERF104, encoding a chaperone protein and an APETELA2/ ethylene-responsive factor, respectively, are strongly induced in a compatible interaction with blast fungus, and also have function in plant susceptibility validated through gene silencing. Here, we reported the CRISPR/Cas9 knockout of OsDjA2 and OsERF104 genes resulting in considerable improvement of blast resistance. A total of 15 OsDjA2 (62.5%) and 17 OsERF104 (70.8%) T 0 transformed lines were identified from 24 regenerated plants for each target and used in downstream experiments. Phenotyping of homozygous T 1 mutant lines revealed not only a significant decrease in the number of blast lesions but also a reduction in the percentage of diseased leaf area, compared with the infected control plants. Our results supported CRISPR/Cas9-mediated target mutation in rice susceptibility genes as a potential and alternative breeding strategy for building resistance to blast disease

Thirty Years of Genome Engineering in Rice: From Gene Addition to Gene Editing

International audienceRice, the staple food for more than half of humankind and the model monocot crop, was the first cereal in which efficient gene transfer procedures were implemented: 30 years ago, the first transgenic rice plant was regenerated following direct gene transfer to cell suspension-derived protoplasts. Shortly thereafter, transgenic plants were regenerated from zygotic embryo-derived cells following their subjection to micro-projectile acceleration and Agrobacterium-mediated transfection treatments. The high efficiency of transfer deoxyribonucleic acid (T-DNA) integration in seed embryo-derived cells of rice has allowed the transfer of genes of agronomical relevance and the generation of large collections of insertion lines and has provided a key contribution to deciphering the function of more than 2000 rice genes. The high efficiency of T-DNA integration in seed embryo-derived cells of rice also permitted the first implementation of gene targeting and knock-in (KI) events, relying on the albeit very low natural frequency of homology-directed repair (HDR) in the rice genome. In the late 2000s, the advent of site-directed nucleases (SDNs) that induce either single or double-strand breaks at a high frequency and their rapid application to rice permitted routine targeted mutagenesis, which can be multiplexed to simultaneously alter several targets or create deletions, and base and gene editing (e.g. correction of amino acids). Currently, the challenge remains to attain a high frequency of KI and replacement of long stretches of DNA for protein domain or coding sequence swapping. We present herein a historical perspective of the advances that have been readily implemented to determine the function of rice genes and to manipulate traits of agronomical relevance. Two main bottlenecks remain to be alleviated in rice genomic engineering: the low frequency of HDR and the genotype dependence of gene transfer efficiency. Alleviation of these bottlenecks is needed to reach the potential of intra- and interspecific gene replacement and SDN-mediated multiplex editing of alleles in elite materials, which will assist in the breeding and deployment of rice cultivars embedded in sustainable and climate-smart agricultural practices

GPX5, the selenium-independent glutathione peroxidase-encoding single copy gene is differentially expressed in mouse epididymis

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Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas9 system. Colloque Inter-LabEx CRISPR-Cas9

Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas9 system. Colloque Inter-LabEx CRISPR-Cas9. Colloque Inter-LabEx CRISPR-Cas