关键词: 拟南芥, CRISPR/Cas9, 基因编辑, 解码, TCH4

Abstract:

【Background】 In recent years, the CRISPR-Cas9 system has been developed to an efficient site-directed gene- editing technology in a variety of plants. It can provide a rapid method for studying gene function. However, the editing efficiency of some CRISPR-Cas9 vectors is very low. 【Objective】 In this study, an efficient CRISPR-Cas9 system, with a red fluorescent protein screening marker and a RIBOSOMAL PROTEIN S5 A (RPS5A) promoter, was constructed to edit the xyloglucan endotransglucosylase/ hydrolase TOUCH4 (TCH4) gene in Arabidopsis thaliana. 【Method】 The CRISPR vector pRSE-WH was constructed from the pKSE401 vector by replacing the 35S promoter with a RPS5A promoter, which maintains high constitutive expression at all developmental stages from the egg cell, and the hygromycin resistance gene with DsRed2. The sgRNA of TCH4 was designed using CRISPR-P 2.0 (http://crispr.hzau.edu.cn). After excluding non-specific target sites by sequence comparisons with the Arabidopsis thaliana genome database, two target sites were selected. The sgRNA oligonucleotides with junctions were chemically synthesized. After annealing, two expression vectors (TCR1 and TCR2, corresponding to the two target sites) were constructed by linking the sgRNA with junctions to the plasmid pRSE-WH respectively. The vectors of TCR1 and TCR2 were transferred into wild-type Col-0 by Agrobacterium-mediated floral-dip transformation. In the T₁ generation, the positive transgenic plants were identified using red fluorescent protein as a marker, and the target sites were detected using PCR combined with Sanger-sequencing to confirm whether it was mutated as expected. The mutations were determined from the peak map of the sequencing results, and the mutation pattern and genotypes were further analyzed. 【Result】 A high efficiency CRISPR vector pRSE-WH for gene editing in Arabidopsis was constructed. The TCR1 and TCR2 expression vectors successfully enabled gene-specific editing of TCH4. In the editing of the TCH4 gene, the mutation ratios using TCR1 and TCR2 were 80%, 100% respectively, and the total editing efficiency was 86%. The mutations in T₁ plants were decoded according to the sequencing results and included homozygous, heterozygous and biallelic mutations. Among 59 mutated T₁ plants, 8 plants (13.56%) were not edited, 9 plants (15.25%) were homozygous, 40 plants (67.80%) were biallelic and 2 plants (3.39%) were heterozygous. The homozygous and biallelic seeds without red fluorescence of T₁ generation were selected for breeding, and sequenced the plants of T₂ generation. The results showed that mutations of the T₁ generation were successfully inherited to T₂ generation, which were Cas9-free. 【Conclusion】 PRSE-WH demonstrated high-efficiency gene editing in Arabidopsis thaliana. It was very easy to obtain Cas9-free and stably inherited mutants in the T₃ generation by screening seeds with red fluorescence marker.

Key words: Arabidopsis thaliana, CRISPR/Cas9, gene editing, decode, TCH4