Oligonucleotide‐directed mutagenesis for precision gene editing

Abstract

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.

New developments in green biotechnology - an inventory for RIVM

“…The synthetic oligo's consist of both DNA and modified nucleotides or other end-protective chemistries. These modifications prevent the oligonucleotides from undergoing recombination (i.e., being incorporated into the genome), while maintaining the ability to act as a mutagen (Sauer et al 2016). Once the correction process is completed, the oligos are degraded.…”

Genome Editing in Plants: An Overview of Tools and Applications

“…TALE proteins consist of a central domain responsible for DNA binding, nuclear localization crRNA, Cas9 proteins Kumar and Jain [6] Sauer et al [7] 2015 2016…”

“…Noman et al [12] 2016 Sauer et al [7] 2016 signal, and a domain that serves as activator of transcription of the target gene (Table 1) [54]. For the first time, the DNAbinding ability of these proteins was described in 2007 [55], and a year later, two scientific groups have decoded the recognition code of target DNA sequence by TALE proteins [56].…”

Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells

“…As an additional option, synthetic chimeric RNA/DNA molecules or single-stranded DNA oligonucleotides (SDOs) can be employed as vectors to produce oligonucleotide-targeted nucleotide exchange (OTNE) at a specific site of plant genomic DNA (Breyer et al 2009;Sauer et al 2016a;Sauer et al 2016b). The pioneering studies preferentially used chimeric RNA/DNA oligonucleotides for the induction of gene-specific mutations (Beetham et al 1999;Kochevenko and Willmitzer 2003;Okuzaki and Toriyama 2004;Ruiter et al 2003;Zhu et al 2000;Zhu et al 1999).…”

“…Out of several parameters, the optimal length and structure of oligonucleotide molecules need to be determined for a given organism. Sauer et al (2016a) reported that oligonucleotide length had a positive correlation with the frequency of targeted edits as monitored by changing the blue fluorescent protein to green fluorescent protein. Dong et al (2006) developed a transient assay system based on cobombardment of mutated GFP plasmid constructs with targeting SDO GFP to the scutellum cells of immature wheat embryos.…”