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

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

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

Psychiatric symptoms and mortality in older adults with major psychiatric disorders: results from a multicenter study

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