Trinh Duy Dinh, Le Ngoc Thu, Bui Thao Phuong, Le Thao Nhu Thi, Nguyen Cuong Xuan, Chu Ha Hoang, Do Phat Tien
Institute of Biotechnology, Vietnam Academy of Science and Technology, Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam.
University of Science and Technology of Hanoi, Vietnam Academy of Science and Technology, Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam.
Saudi J Biol Sci. 2022 Oct;29(10):103420. doi: 10.1016/j.sjbs.2022.103420. Epub 2022 Aug 17.
This study was performed to evaluate the sequential transformation for soybean genome editing using the CRISPR/Cas9 system as well as to show a strategy for examining the activity of CRISPR/Cas9 constructs, especially the designed guide RNAs (gRNAs). The gRNAs for targeted mutations of an exogenous gene and multiple endogenous genes were constructed and transferred into a stably-overexpressed-Cas9 soybean line using -mediated hairy root induction system. The targeted mutations were identified and characterized by the poly-acrylamide gel electrophoresis (PAGE) heteroduplex method and by sequencing. Induced mutations of the exogenous gene () were observed in 57% of tested transgenic hairy roots, while 100% of the transgenic root lines showed targeted mutations of the endogenous ) gene. Multiple gRNAs targeting two endogenous genes ( and ) induced mutation rates of 75% and 67%, respectively. Various indels including small and large deletions as well as insertions were found in target sites of the tested genes. This sequential transformation method could present the targeting efficacy of different gRNAs of each tested gene. Additionally, in this study differences in gRNA ratings were found between bioinformatics predictions and actual experimental results. This is the first successful application of the sequential transformation method for genome editing in soybean using the hairy root system. This method could be potentially useful for validating CRISPR/Cas9 constructs, evaluating gRNA targeting efficiencies, and could be applied for other research directions.
本研究旨在评估使用CRISPR/Cas9系统对大豆基因组进行编辑的连续转化方法，并展示一种检测CRISPR/Cas9构建体活性的策略，特别是设计的引导RNA（gRNA）。构建了用于靶向突变外源基因和多个内源基因的gRNA，并使用发根诱导系统将其转入稳定过表达Cas9的大豆品系中。通过聚丙烯酰胺凝胶电泳（PAGE）异源双链法和测序对靶向突变进行鉴定和表征。在57%的测试转基因毛状根中观察到外源基因（）的诱导突变，而100%的转基因根系显示出内源（）基因的靶向突变。靶向两个内源基因（和）的多个gRNA的诱导突变率分别为75%和67%。在测试基因的靶位点发现了各种插入缺失，包括小的和大的缺失以及插入。这种连续转化方法可以呈现每个测试基因不同gRNA的靶向效率。此外，在本研究中，发现生物信息学预测与实际实验结果之间的gRNA评级存在差异。这是首次成功应用连续转化方法通过毛状根系统对大豆进行基因组编辑。该方法可能有助于验证CRISPR/Cas9构建体、评估gRNA靶向效率，并可应用于其他研究方向。
