Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 59-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants.

SummaryDifferences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide‐directed mutagenesis (ODM), one of the many tools of Cibus’ Rapid Trait Development System (RTDS ™) technology, offers a rapid, precise and non‐transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide‐mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.

Shigella move through the cytosol of infected cells by assembly of a propulsive actin tail at one end of the bacterium. Vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins, is important in cellular actin dynamics and is present on intracellular Shigella. VASP binds both profilin, an actin monomer-binding protein, and vinculin, a component of intercellular contacts that also binds the Shigella actin assembly protein IcsA. It has been postulated that VASP might serve as a linker between vinculin and profilin on intracellular Shigella, thereby delivering profilin to the Shigella actin assembly machinery. We show that Shigella actin-based motility is unaltered in cells that are deficient for the Ena/VASP family of proteins. In these cells, Shigella form normal-appearing actin tails and move at rates that are comparable to the rates of bacterial movement in Ena/VASP-deficient cells complemented with the Ena/VASP family member Mena. Finally, whereas vinculin can bind the Arp2/3 complex, we show that Arp2/3 recruitment to Shigella is not correlated with vinculin recruitment, indicating that the role of vinculin in Shigella motility is not recruitment of Arp2/3. Thus, although VASP is recruited to the surface of intracellular Shigella, it is not essential for Shigella actin-based motility.