关键词: 水稻, CRISPR/Cas9, 基因编辑, OsPIL15, 脱靶效应

Abstract: 【Objective】 As an important environmental signal, light can regulate gene expression, affect the activity of enzymes and plant morphogenesis. Phytochrome interacting factors play an important role in the signal transduction of light. Therefore, constructing the expression vector of CRISPR/Cas9 containing rice phytochrome-interacting factors OsPIL15 and creating the ospil15 mutants have important significance to exploit functional genes and enrich light signal regulation of the molecular mechanism of rice. 【Method】 According to the principle of CRISPR/Cas9 technology, the sgRNA was designed. To exclude non-specific target sites, the sgRNA was analyzed by sequence alignment in the rice genome database. At the same time, the target sequence contained the common restriction site to identify mutants. The oligonucleotides of sgRNA were chemically synthesized and inserted into linearized plasmid pBUN411 to construct the expression vector. Transgenic rice plants harboring sgRNA:Cas9 were obtained by A. tumefaciens-mediated stable transformation and the positive transgenic plants were screened by herbicide resistance. The PCR products of T₀ transgenic plants were digested by restriction enzyme to judge whether they were mutants. Then, the mutated genotypes of these mutants were analyzed by DNA sequencing. After searching rice genome using sgRNA sequence, five highly identical sites with less 4 mismatching bases were selected to assess off-target efficiency and specificity of sgRNA.【Result】The recombinant vector succeeded in oriented editing of OsPIL15. The restriction enzyme analysis results indicated that 15 mutants from the 25 randomly selected T₀ transgenic lines were obtained. They included 5 homozygous mutants, 6 biallelic mutants and 4 heterozygous mutants, and a total of 10 different genotypes and 11 mutant lines. The mutant types were mainly insertions or deletions of single base, besides, two mutant types of large deletions with 56 and 66 bp were obtained. Analysis of T₁ transgenic plants from some T₀ mutants indicated that the genotypes in T₀ mutants could descend stably into the next generation. The progeny of homozygous mutants in T₀ were homozygous mutants. However, only one wild type with no mutation was detected in a homozygous mutant progenies of line 14. Two homozygous mutations and one biallelic mutations were obtained in the progeny of biallelic T₀ lines. Three mutation types including homozygous mutants, heterozygous mutants and wild type with no mutation were detected in the progenies of heterozygous T₀ mutants. Restriction enzyme analysis was used to detect the engineered target site of T₁ positive transgenic plants which had no mutation in T₀. No mutation in 62 T₁ plants was detected. The results showed that CRISPR/Cas9 system played no role in gene editing in T₁ positive transgenic plants. After searching the rice genome using the target sequence with PAM, five highly identical sites were found. However, any mutations at these sites in T₀ and T₁ generations were not observed by restriction enzyme, which indicated the sgRNA was highly specific. Three groups of different genotypes were selected representative ospil15 mutants in T₁ generation to observe phenotypes, the investigation result showed that the growth stage and tiller number were not obviously changed, the plant height decreased significantly (P＜0.01), and the grain length was significantly increased compared with the wild type, up 5.69%. 【Conclusion】CRISPR/Cas9 system succeeded in oriented editing of OsPIL15, the ospil15 mutants with 10 different genotypes were obtained and observed that the plant height decreased significantly and the grain length increased significantly compared with those of wild type.

Key words: rice, CRISPR/Cas9, gene editing, OsPIL15, off-target efficiency