Gossypium hirsutumis an allotetraploid species, meaning that mutants that are difficultto be generated by classical approaches due to gene redundancy. The CRISPR/Cas9genome editing system is a robust and highly efficient tool for generating target genemutants, by which the genes of interest may be functionally dissected and applied through genotype-to-phenotype approaches. In this study, the CRISPR/Cas9 genomeediting system was developed inG. hirsutumthrough editing theGh14-3-3dgene. In T0 transgenic plants, lots of insertions and deletions (indels) inGh14-3-3dat the expected target site were detected in the allotetraploid cotton At or Dt subgenomes. The results

of the PCR, T7EI digestion and sequencing analyses showed that the indels inGh14-3-3dgene can be stably transmitted to the next generation. Additionally, the indelsin the At and Dt subgenomes were segregated in the T1 transgenic plants followingMendelian law, independing on the T-DNA segregation. Two homozygousGh14-3-3deditedplants free of T-DNA were chosen by PCR and sequencing assays in the T1plants, which were called transgene-clean editing plants and were designatedce1andce2in the T2 lines showed higher resistance toVerticillium dahliaeinfestation comparedto the wild-type plants. Thus, the two transgene-clean edited lines can be used as agermplasm to breed disease-resistant cotton cultivars, possibly avoiding complex andexpensive safety assessments of the transgenic plants.