Oligonucleotide-Mediated Genome Editing in Plants

Researchers from CIBUS in the US report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants. This form uses single-stranded oligonucleotides (ssODNs) to generate genome edits at DNA strand lesions made by DNA double strand break reagents.

Using Arabidopsis, the team obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts 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 through TALENs or CRISPR/Cas9, resulted in a significantly greater targeted genome editing frequency compared with treatment with DNA double strand-breaking reagents alone.

The researchers then tested a combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerant flax (Linum usitatissimum) by editing the 5'-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. The edits occurred at sufficient frequency that the team could regenerate whole plants from edited protoplasts without selection. Plants were screened for tolerance to glyphosate in spray tests. Analysis of their progeny reveals the expected Mendelian segregation of the EPSPS edits.

For more information, read the full article in Plant Physiology.