关键词: CRISPR-Cas9, 拟南芥, 基因编辑, 酶切鉴定, SDP1

Abstract: Nowadays, the technology for site-specific editing of plants using the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system has been matured,but the identification of the progeny of the edited plant is still time-consuming and labor-intensive.In order to optimize the identification method,we took Arabidopsis sugar-dependent 1(SDP1)gene as the editing object.Sequence analysis showed that there was protospacer adjacent motif(PAM)in exons of SDP1 gene,and there were four suitable restriction sites in upstream of PAM sequences.We screened these restriction sites for their positions and performance,selected the restriction site Sac Ⅰ in the second exon of SDP1 gene as the editing target,and constructed the CRISPR-SDP1-Cas9 vector.Through enzyme digestion identification and sequencing analysis of T1 seedlings screened with hygromycin,the heterozygous edited lines with three bands were identified.Among 71 randomly selected T1 plants,24 heterozygous edited lines were found,the editing efficiency was 16.9%.Among 48 T2 plants,5 homozygous edited lines were screened out,and the editing sites were all insertions at the Sac Ⅰ restriction site.In summary, the appropriate restriction sites near the PAM sequence of the gene to be edited were selected as editing targets,and the progeny plants were identified through the restriction enzyme digestion and sequencing analysis.The traditional method identifing the progeny of CRISPR-Cas9 transgenic plants is extracting DNA from the progeny plants,amplifing the target site by PCR and connecting it to the T vector for sequencing several times.This method is simple,fast,economical and practical compared with the traditional method,providing a simple and quick method for CRISPR-Cas9 gene-edited progeny identification.

Key words: CRISPR-Cas9, Arabidopsis thaliana, Gene editing, Restriction enzyme digestion identification, SDP1