A sequential transformation method for validating soybean genome editing by CRISPR/Cas9 system

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 Agrobacterium rhizogenes-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 (gus) were observed in 57% of tested transgenic hairy roots, while 100% of the transgenic root lines showed targeted mutations of the endogenous (SACPD-C) gene. Multiple gRNAs targeting two endogenous genes (SACPD-C and SMT) 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.