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The CRISPR-Cas9-based multiplexed gene editing (MGE) provides a powerful method to modify multiple genomic regions simultaneously controlling different agronomic traits in crops. We applied the MGE construct built by combining the tandemly arrayed tRNA-gRNA units to generate heritable mutations in the TaGW2 , TaLpx-1 , and TaMLO genes of hexaploid wheat. The knockout mutations generated by this construct in all three homoeologous copies of one of the target genes, TaGW2 , resulted in a substantial increase in seed size and thousand grain weight. We showed that the non-modified gRNA targets in the early generation plants can be edited by CRISPR-Cas9 in the following generations. Our results demonstrate that transgenerational gene editing activity can serve as the source of novel variation in the progeny of CRISPR-Cas9-expressing plants and suggest that the Cas9-inducible trait transfer for crop improvement can be achieved by crossing the plants expressing the gene editing constructs with the lines of interest.

Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat