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.
Figure 2: Gene-targeting of MS8 gene in male sterile F2 plants. (A) Diagram of the MS8 gene and the target site; four exons were numbered; the PAM motif is underlined and in bold. (B) The sequencing chromatograms of MS8 in maize inbred line B73, ms8-DelG/ms8-DelG, and ms8-InA/ms8-InA plants. T0 transgenic line H17 plant was crossed with maize inbred line Zong31 to produce F1 generation, which was then self-pollinated to produce F2 progeny. Fragments flanking the target site were amplified by PCR using the genomic DNA of male sterile F2 plants as the template, and the PCR products were directly sequenced.