Development and Application of a Codon-Optimized CRISPR/Cas9 System for Targeted Mutagenesis in Soybean and Medicago truncatula

With rapid technological advances in the past two years, the CRISPR/Cas9 system has become an efficient and valuable tool for gene editing in higher plants. Technical optimization includes the testing of codon-optimized Cas9 nuclease genes in combination with different promoters to obtain high expression levels in transgenic plant tissues.  Since targeted mutagenesis still depends on transgenesis, an efficient delivery/transformation system is essential for assessing the full potential of the CRISPR/Cas9 system, especially in recalcitrant crops such as soybean. Our design of the CRISPR/Cas9 system involves the utility of a soybean codon-optimized Cas9 coding region driven by various promoters, incorporation of a constitutively-expressed GUS reporter gene, and a medium-free hairy root induction system. Our initial results showed that mutations were generated efficiently in both soybean and the legume model species Medicago truncatula. Furthermore, analysis of the mutations revealed that microhomology-mediated end-joining occurs frequently in the repair of double-stranded breaks generated by the Cas9 nuclease in soybean. Depending on the genomic structure of the target genes, microhomologous sequences flanking the target site could serve as “hotspots” for generating precise deletions between microhomologous mini-repeats.