Efficient, selectable marker free gene targeting in soybean using novel Ochrobactrum haywardense -mediated delivery

作者：Sandeep Kumar, Zhan-Bin Liu, Nathalie Sanyour-Doyel, Brian Lenderts, Andrew Worden, Ajith Anand, Hyeon‐Je Cho, Joy Bolar, C. Jake Harris, Lingxia Huang, Aiqiu Xing, Alexandra Richardson · 发表于：bioRxiv (Cold Spring Harbor Laboratory) · 年份：2021 · DOI：10.1101/2021.10.28.466326 · 研究领域：CRISPR and Genetic Engineering、Plant tissue culture and regeneration、Chromosomal and Genetic Variations

Abstract We report robust selectable marker-free gene targeting (GT) system in soybean, one of the most economically important crops. A novel efficient Ochrobactrum haywardense -mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants in 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 a need of a selectable marker within the template. High-resolution Southern by Sequencing (SbS) 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 selectable marker within the donor DNA, and generates non-chimeric plants with heritable GT.