Objective To obtain transgene-free progeny by constructing a DNA editing system with a fluorescent screening marker gene and two pairs of single-guide RNAs to simultaneously recognize two different sites in the target gene encoding Arabidopsis microRNA(miR)160A Results The T-1 seeds with red fluorescence were easily identified and were selected to verify that the proportion of miR160A knockout mutants reached approximately 50%. Seeds with no fluorescence in the T-2 generation were selected and screened for homozygous mutants. In the T-2 generation plants, the Cas9 fragment was not detected by polymerase chain reaction. The traits of the homozygous mutants were stably inherited by the T-2 population. Conclusions A highly efficient DNA-editing construct was successfully developed and can be used as a plant genome site-specific editing tool that may be useful for improving plant genetic resources.