关键词: CRISPR/Cas9, 基因编辑, 荧光筛选标记, as1

Abstract: The CRISPR/Cas9 gene-editing system is easy to handle and has a high gene editing efficiency. However, how to quickly screen and obtain gene editing descendants without exogenous transformation elements is a key technical problem. In this study, two pairs of single-guide RNAs were designed to simultaneously recognize two different sites in the one target gene encoding as1 in Arabidopsis. The CRISPR construct also carried a gene for a fluorescent selection marker. By fluorescence screening, a transgene-free progeny was easily obtained in the T₂ population. T₁ seeds with red fluorescence in their coat were selected to verify that the proportion of as1 knockout mutants traits were obvious. Seeds with no fluorescence were selected among the T₁ generation and screened for homozygous mutations. In the T₂ generation plants, the Cas9 fragment was not detected by polymerase chain reaction. Thus, a highly efficient DNA-editing construct, with a gene for a fluorescence screening marker, was successfully constructed, and the transgenic element was successfully eliminated from the progeny by selecting or avoiding seeds with the fluorescent marker. This system can be used as a plant genome site-specific editing tool and may be useful for improving plant genetic resources.

Key words: CRISPR/Cas9, gene editing, fluorescence screening, as1