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.

CsV03-3 is a member of a novel gene family from citrus that encodes a protein with DNA binding activity and whose expression is responsive to defense signals and abiotic stress.

We have identified a novel gene designated CsV03-3 from Citrus sinensis (L.) Osbeck that encodes a 50 amino acid polypeptide with a predicted molecular mass of 5.4kDa and a pI of 3.7. CsV03-3 expression is up-regulated by application of methyl jasmonate, salicylic acid, and abscisic acid as well as by abiotic stress and insect herbivory. CsV03-3 belongs to a small gene family consisting of at least three other closely related members (CsV03-1, CsV03-2, and CsV03-4) whose expression is also responsive to phytohormone application and abiotic stress. Sequence similarity searches of the public databases were unsuccessful in finding sequence homologs to CsV03-3 or any CsV03 family member; however, structural prediction models coupled with model comparison to Protein Data Bank folds indicated that the predicted polypeptide encoded by CsV03-3 has structural similarity to proteins with nucleic acid binding activity. Gel mobility shift assays performed on recombinant CsV03-3 protein demonstrated active binding with dsDNA and, to a lesser extent, ssDNA. Based on the phytohormone-inducible expression patterns, the ability to bind nucleic acids, and the lack of significant sequence homology to public databases, we propose that CsV03-3 and its related homologs defines a new class of nucleic acid binding proteins that are responsive to defense and stress signaling in woody perennials.

Genome sequence and molecular characterization of Homalodisca coagulata virus-1, a novel virus discovered in the glassy-winged sharpshooter (Hemiptera: Cicadellidae).

The complete nucleotide sequence of a novel single-stranded RNA virus infecting the glassy-winged sharpshooter, Homalodisca coagulata, has been determined. In silico analysis of H. coagulata virus-1 (HoCV-1) revealed a 9321-nt polyadenylated genome encoding two large open reading frames (ORF1 and ORF2) separated by a 182-nt intergenic region (IGR). The deduced amino acid sequence of the 5'-proximal ORF (ORF1, nt 420-5807) exhibited conserved core motifs characteristic of the helicases, cysteine proteases, and RNA-dependent RNA polymerases of other insect-infecting picorna-like viruses. A structural model created using Mfold exposed a series of stem loop (SL) structures immediately preceding the second ORF which are analogous to an internal ribosome entry site (IRES), suggesting that ORF2 begins with a noncognate GCA triplet rather than the canonical AUG. This 3' ORF2 (5990-8740) showed significant similarity to the structural proteins of members of the family Dicistroviridae, particularly those belonging to the genus Cripavirus. Evidence demonstrating relatedness of these viruses regarding genome organization, amino acid sequence similarity, and putative replication strategy substantiate inclusion of HoCV-1 into this taxonomic position.