Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) f lanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green f luorescent protein transgene that had been designed to contain a single base deletion. Recovery of f luorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.Genomics is currently a central component of plant biology research. The gene sequence of Arabidopsis thaliana will soon be available, and many other species are under study. Identification of new genes is occurring at a much faster pace than is the determination of their function. One missing technology in plant biology is the ability to selectively and reliably create site-specific ''gene knockouts'' or homologous recombination of genes of interest or of unknown function. The phenomena of gene silencing (e.g., antisense and cosuppression) provides a method for understanding gene function through the creation of transgenic crops expressing a gene sequence that silences the endogenous gene. However, this approach is not suitable for functional genomic studies of individual members of multigene families or genes with similar sequence, and is sometimes problematic in other technical aspects (1).A technology currently being explored in prokaryotic and eukaryotic systems uses self-complementary chimeric oligonucleotides (COs) comprised of DNA and 2Ј-O-methyl RNA to target and mutate genes in vivo. These COs are designed to have one or more bases that do not pair with the endogenous gene sequence. This approach was successfully used to modify endogenous genes of mammalian cells (2-5) in a site-specific and genetically inheritable manner. Recently, Alexeev and Yoon (5) have demonstrated that permanent in vivo conversions result in phenotypic changes in mouse melanocytes. It has been hypothesized that the mechan...

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.

Oligonucleotide-directed gene repair is a potential technique for agricultural trait modification in economically important crops. However, large variation in the repair frequencies among the scientific reports indicates that there are many factors influencing the repair process. We report here a transient assay system using GFP as a reporter for testing the efficiency of plasmid DNA repair in cultured wheat cells. This assay showed that osmotic medium supplemented with 2,4-D increased the oligo-targeting frequency, and that the repair of a point mutation was more efficient than repair of a single base deletion mutation in cultured scutellum cells of immature wheat embryos. This study provides the first evidence that oligonucleotide-directed mutagenesis is applicable to regenerable cultured wheat scutellum cells.

We have mapped the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1. Northern hybridization and 3h RACE analysis identified two polyadenylated RNAs associated with BBTV DNA-1. Previously, one major ORF in the virion sense of DNA-1 had been identified, which encoded a putative replication protein (Rep). An mRNA was identified in BBTV infected bananas that was clearly transcribed from this Rep ORF. Further, a second transcript was identified which mapped to an ORF completely within the Rep ORF. This encoded a putative 5 kDa protein of unknown function. Both these transcripts were also identified in a tobacco plant that had been transformed with Agrobacterium tumefaciens harbouring a binary construct containing the Rep ORF from BBTV DNA-1. This Rep ORF

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Promoter regions associated with each of the six ssDNA components of banana bunchy top virus (BBTV) have been characterized. DNA segments incorporating the intergenic regions of BBTV DNA-1 to -6 were isolated and fused to the uidA (β-glucuronidase) reporter gene to assess promoter activity. In tobacco cell suspensions, the BBTV DNA-2 and -6 promoters generated levels of GUS expression 2-fold greater and similar to the 800 bp CaMV 35S promoter, respectively. Deletion analysis of the BBTV DNA-6 promoter suggested all the necessary promoter elements required for strong expression were located within 239 nucleotides upstream of the translational start codon. In transgenic tobacco plants, the BBTV-derived promoters generally provided a weak, tissue-specific GUS

Banana bunchy top virus (BBTV) DNA-3 to 6 have each previously been shown to contain one large open reading frame in the virion sense, whereas no large ORF had been identified in BBTV DNA-2. RNAs transcribed from the BBTV genome were mapped using northern hybridisation and 3' RACE. One mRNA was transcribed from each of BBTV DNA-2 to 6 and four of these mRNAs mapped to the ORFs previously identified in BBTV DNA-3 to 6. The mRNA of BBTV DNA-2 was transcribed from a virion sense ORF probably using a TATA box sequence different to that in BBTV DNA-1, and DNA-3 to 6. This ORF encoded a 10 kDa protein of unknown function. The 3' untranslated region of the five mRNAs varied from 25 nucleotides (BBTV DNA-6) to 167 nucleotides (BBTV DNA-4) and each contained putative polyadenylation signals with associated GT rich sequence together with a possible termination signal (C/T/A)TGTAA conserved in all five mRNAs.