An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice

• Published in: International journal of molecular sciences 2022-01, Vol.23 (3), p.1588
• Main Authors: Tan, Jiantao, Wang, Yaxi, Chen, Shuifu, Lin, Zhansheng, Zhao, Yanchang, Xue, Yang, Luo, Yuyu, Liu, Yao-Guang, Zhu, Qinlong
• Format: Article
• Language: English
• Subjects: Antibiotics; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; DNA damage; DNA repair; DNA Shuffling; Efficiency; Flowers - genetics; Flowers - growth & development; Gene deletion; Gene Editing - methods; Gene expression; Genes; Genetic engineering; Genetic transformation; Genome editing; Genomes; Homologous Recombination; Homology; homology-directed repair; marker-free; Oryza - genetics; Oryza - growth & development; Plant Proteins - genetics; Plant Shoots - genetics; Plant Shoots - growth & development; Plant Stems - genetics; Plant Stems - growth & development; Progeny; Promoter Regions, Genetic; Repair; rice; stem-, shoot tip- and inflorescence-strong promoter (Pssi); Transformed cells; Transgenic plants
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23031588

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (P ) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this P to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T plants and 83.2% of T plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.