A DNA-free and genotype-independent CRISPR/Cas9 system in soybean

Here, we report a smart genome editing system for soybean (Glycine max) using the in planta bombardment-ribonucleoprotein (iPB-RNP) method without introducing foreign DNA or requiring traditional tissue culture processes such as embryogenesis and organogenesis. Shoot apical meristem (SAM) of embryonic axes was used as the target tissue for genome editing because the SAM in soybean mature seeds has stem cells and specific cell layers that develop germ cells during the reproductive growth stage. In the iPB-RNP method, the RNP complex of the CRISPR/Cas9 system was directly delivered into SAM stem cells via particle bombardment, and genome-edited plants were generated from these SAMs. Soybean allergenic gene Gly m Bd 30K was targeted in this study. Many E0 (the first generation of genome-edited) plants in this experiment harbored mutant alleles at the targeted locus. Editing frequency of inducing mutations transmissible to the E1 generation was approximately 0.4% to 4.6% of all E0 plants utilized in various soybean varieties. Furthermore, simultaneous mutagenesis by iPB-RNP method was also successfully performed at other loci. Our results offer a practical approach for both plant regeneration and DNA-free genome editing achieved by delivering RNP into the SAM of dicotyledonous plants.