CRISPR/Cas9 Transformation of Maize

The CRISPR/Cas9 binary vector pBUE-2gRNA-ZH was transformed into Agrobacterium strain EHA105, and Agrobacterium-mediated method was used to transform immature embryos of B73 maize at China Agricultural University Transgenic Facility Center. The genomic DNA was extracted from 20 transgenic seedlings and the PCR fragment, primers and reactions were the same as those described above. For restriction enzyme digestion analysis, about 500 ng purified PCR products from each reaction was digested overnight with XcmI or SphI in a 20-μL reaction volume. For sequencing analysis, the PCR products from two representative transgenic seedlings were cloned into the cloning vector pCBC and positive clones were sequenced using the T7 primer.