Streamlined Agrobacterium rhizogenes-mediated hairy root transformation for efficient CRISPR/Cas9-based gene editing evaluation in diverse Citrullus cultivars

作者：Yong Zhao, Hongju Zhu, Xuqiang Lü, Muhammad Anees, Nan He, Dongdong Yang, Zihao Chen, Zonglie Hong, Junhong Zhang, Wenge Liu · 发表于：Horticultural Plant Journal · 年份：2024 · DOI：10.1016/j.hpj.2023.10.005 · 被引用次数：20 · 研究领域：CRISPR and Genetic Engineering、Plant tissue culture and regeneration、Chromosomal and Genetic Variations

The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics. Within this system, single guide RNAs (sgRNAs) are instrumental in the precise cleavage of DNA double strands. However, the efficiency of gene editing varies among sgRNAs, emphasizing the need to meticulously select target sites, especially in the context of Citrullus lanatus, a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus, Citrullus mucosospermus, and Citrullus amarus. Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four Citrullus species. Notably, 73.94% of all examined hairy roots exhibited mutations at the sgRNA1 site, while the sgRNA5 site showed no mutations. Among the 31 different mutation types identified at the sgRNA1 site, base deletion was the most prevalent. Using the sgRNA1 site of ClCIPK17, stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of th...