CRISPR-Enabled Complex Trait Loci for Gene Stacking in Maize

Sustainable maize production requires combining multiple biotech and native traits that provide insect control, disease resistance and tolerance to herbicides or abiotic stresses. Because trait genes have been randomly inserted at various genomic locations, combining numerous traits is expensive, time-consuming and complex. Here we report the Complex Trait Locus (CTL) for genetic trait stacking. CTL consisted of multiple preselected sites which were positioned within a small, well characterized chromosomal region. Firstly, a site-specific insertion landing pad (SSILP) was targeted to one of the sites using CRISPR-Cas9 technology, producing individual inbred lines capable of receiving trait genes via recombinase-mediated cassette exchange (RMCE). The traits then can be combined within a linkage block using genetic crossing and bred as a single locus. We generated 57 SSILP lines at 4 CTLs across 3 inbreds. The NPTII gene in the SSILP expressed consistently and had minimal impact on neighboring endogenous gene expression. Two-gene stacking was readily achieved. The CTL technology is a major step forward in streamlining biotech product testing and deployment. This study also advances our understanding of gene targeting and expression.