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 , a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an -mediated hairy root method to assess effective target sites for gene editing of across various species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of and . Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four 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 , stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse species.

中文翻译：

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简化发根农杆菌介导的毛状根转化，可在多种西瓜品种中进行基于 CRISPR/Cas9 的高效基因编辑评估

CRISPR/Cas9 基因组编辑系统是增强作物遗传学的关键工具。在此系统中，单引导 RNA (sgRNA) 在 DNA 双链的精确切割中发挥着重要作用。然而，基因编辑的效率因 sgRNA 的不同而不同，强调需要精心选择靶位点，特别是在这种因通过稳定转化产生转基因植物具有挑战性和低效而臭名昭著的物种的背景下。这项研究采用介导的毛根方法来评估不同物种基因编辑的有效靶位点。在 和 的不同生长阶段的不同植物组织中成功诱导了毛状根。使用位于 CRISPR/Cas9 系统的外显子 1 和外显子 5 保守区域内的两个 sgRNA（sgRNA1 和 sgRNA5）的载体，在四个物种中 90.9% 的种质中检测到了靶向突变。值得注意的是，在所有检查的毛状根中，73.94% 在 sgRNA1 位点表现出突变，而 sgRNA5 位点没有显示突变。在 sgRNA1 位点发现的 31 种不同突变类型中，碱基缺失最为普遍。利用 的sgRNA1位点，从外植体中获得了稳定的转基因西瓜芽，并证实了sgRNA1位点的靶向突变。这些发现强调了毛根转化系统在评估不同物种中 sgRNA 靶标的编辑效率方面的可行性。