RNA‐guided Cas9 as an <i>in vivo</i> desired‐target mutator in maize

The RNA-guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof-of-concept. Our system showed 51.5%-91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%-28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM-generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM-generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivoDTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof-of-concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA-guided Cas9 genome editing.