Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants.

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 5'-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.

Oligonucleotide-directed mutagenesis for precision gene editing.

Differences 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.

Early increased expression of a cyclin-dependant protein kinase (LtCDKA1;1) during inflorescence initiation of the long day grass Lolium temulentum.

Knowing where and when different genes express at the shoot apex during the transition to flowering will help in understanding this developmental switch. The CDKA family of serine/threonine kinase genes are appropriate candidates for such developmental switching as they are involved in the regulation of the G1/S and G2/M boundaries of the cell cycle (see review by Dudits et al. 2007) and so could regulate increases of cell division associated with flowering. Furthermore, in rice stems the gibberellin (GA) class of plant growth regulators rapidly upregulate CDKA expression and cell division. Thus, CDKA expression might be linked to the florigenic action of GA as a photoperiodically-generated, signal. For the grass Lolium temulentum L., we have isolated an LtCDKA1;1 gene, which is upregulated in shoot apices collected soon after the start of a single florally inductive long day (LD). In contrast to weak expression of LtCDKA1;1 in the vegetative shoot apex, in situ and PCR-based mRNA assays and immunological studies of its protein show very rapid increases in the apical dome at the time that florigenic signals arrive at the apex (<6h after the end of the LD). By ~54h LtCDKA1;1 mRNA is localised to the floral target cells, the spikelet primordia. Later both LtCDKA1;1 mRNA and protein are most evident in floret meristems. Only ~10% of cells within the apical dome are dividing at any time but the LD increase in LtCDKA1;1 may reflect an early transient increase in the mitotic index (Jacqmard et al. 1993) as well as a later increase when spikelet primordia form. Increased expression of an AP1-like gene (LtMADS2) follows that of LtCDKA1;1. Overall, LtCDKA1;1 is a useful marker of both early florigenic signalling and of later morphological/developmental aspects of the floral transition.