A Comparison of ta-siRNA and CRISPR/Cas9 Mediated Gene Silencing Systems in Soybean

Techniques to modify gene expression in vivo are important for understanding gene function and developing novel agronomic traits.  In order to use a ta-siRNA gene-silencing system, the miRNA recognition sequences must be known.  A small RNA dataset was assembled to the soybean genome and nine loci with phased clusters of siRNAs with six unique, 22-nt miRNA recognition sequences were identified.  When these miRNA recognition sequences were fused to a transgenic target sequence, siRNAs were produced to that target sequence, which led to silencing in 95% of hairy root events.  By contrast, CRISPR/Cas systems permanently eliminate gene expression by modifying the target gene sequence.  Using the CRISPR/Cas system to knock out the same genes in soybean made it evident that the frequency of knockout can vary between targets.  One targeting vector knocked out 88% of the events, whereas another target only produced knockouts 18% of the time.  Both ta-siRNA and CRISPR/Cas vectors are simple to construct, effective at modifying gene expression, and offer a number of advantages over conventional systems.  Different types of non-specific effects are possible with both systems, but such off-targeting can be predicted and likely avoided.  Each method is effective and efficient at modifying gene expression, but given the different modes of action, the use of one system versus the other depends on the intended use.