Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template

A gene targeting (GT) method shows 2%-3.4% targeted insertion of the selectable marker-free donor in two different soybean genomic sites. Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with similar to 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

Automated summary

This study reports a selectable marker-free gene targeting system in soybean, which is fast, efficient, reproducible, and capable of generating nonchimeric plants with heritable gene targeting. This system is of great significance in soybean precision breeding.