Generation of Transgene-Free Maize Male Sterile Lines Using the CRISPR/Cas9 System

Male sterility (MS) provides a useful breeding tool to harness hybrid vigor for hybrid seed production. It is necessary to generate new male sterile mutant lines for the development of hybrid seed production technology. The CRISPR/Cas9 technology is well suited for targeting genomes to generate male sterile mutants. In this study, we artificially synthesized Streptococcus pyogenes Cas9 gene with biased codons of maize. A CRISPR/Cas9 vector targeting the MS8 gene of maize was constructed and transformed into maize using an Agrobacterium-mediated method, and eight T₀ independent transgenic lines were generated. Sequencing results showed that MS8 genes in these T₀ transgenic lines were not mutated. However, we detected mutations in the MS8 gene in F₁ and F₂ progenies of the transgenic line H17. A potential off-target site sequence which had a single nucleotide that was different from the target was also mutated in the F₂ progeny of the transgenic line H17. Mutation in the MS8 gene and the male sterile phenotype could be stably inherited by the next generation in a Mendelian fashion. Transgene-free ms8 male sterile plants were obtained by screening the F₂ generation of male sterile plants, and the MS phenotype could be introduced into other elite inbred lines for hybrid production.