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

Abstract

The CRISPR/Cas9 system has revolutionized the field of plant genetic engineering. Here we report a smart genome editing system of soybean by using iPB-RNP method without introducing foreign DNA and requiring traditional tissue culture processes such as embryogenesis and organogenesis. Shoot apical meristem (SAM) of embryonic axes was used the target tissue for genome editing, because the SAM in soybean mature seeds has stem cells and specific cell layer developing germ cells during reproductive growth stage. In the iPB-RNP method, the complex of ribonucleoprotein (RNP) and Cas9 protein 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, which we previously generated genome-editing soybean by using Agrobacterium-mediated transformation and particle bombardment in our previous studies, 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, Gly m Bd 30K protein in mature seeds was not detected by western blot analysis due to flame-sift mutations. 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.