{Reference Type}: Journal Article
{Title}: Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.
{Author}: Kumar S;Liu ZB;Sanyour-Doyel N;Lenderts B;Worden A;Anand A;Cho HJ;Bolar J;Harris C;Huang L;Xing A;Richardson A;
{Journal}: Plant Physiol
{Volume}: 189
{Issue}: 2
{Year}: 06 2022 1
{Factor}: 8.005
{DOI}: 10.1093/plphys/kiac075
{Abstract}: 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 ∼ 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.