Translating Targeted Mutagenesis Technologies into Crop Species

Several methodologies have recently been developed to target specific DNA modifications within a genome.  Researchers in a wide range of crop species are developing knockout or gene deletions at specific loci for desirable trait outcomes. This talk will address recent advances in developing targeted mutagenesis systems in soybean and Medicago.  Soybean has seven functional dicer-like (Dcl) genes as a result of past duplication events, including a single-copy Dcl3 locus and two copies each of Dcl1, Dcl2 and Dcl4. These genes function in RNA processing and are critical for proper RNA silencing and regulation of gene expression. To study their specific (and potentially redundant) roles in the RNA silencing pathways, we have targeted each soybean Dcl gene with either a zinc-finger nuclease (ZFN), a TAL-effector nuclease (TALEN) or a CRIPSR/Cas9 nuclease designed to induce mutations. To date, we have generated non-transgenic lines harboring mutations for five of the seven Dcl genes. In addition, we have used the same platform to engineer Medicago mutants to validate candidate genes associated with symbiotic nitrogen fixation (SNF) previously identified by genome wide association studies (GWAS). This work demonstrates the capacity to develop novel mutant alleles though targeted mutagenesis and derive new phenotypes through stacking these alleles. These efforts have also provided insight into the opportunities and potential obstacles that exist in the translation of gene targeting tools into crop species.