Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat

Authors: WANG, WEI - KANSAS STATE UNIVERSITY; PAN, QIANLI - KANSAS STATE UNIVERSITY; HE, FEI - KANSAS STATE UNIVERSITY; AKHUNOVA, ALINA - KANSAS STATE UNIVERSITY; CHAO, SHIAOMAN; TRICK, HAROLD - KANSAS STATE UNIVERSITY; AKHUNOV, EDUARD - KANSAS STATE UNIVERSITY

Submitted to: The CRISPR Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/21/2018
Publication Date: 2/1/2018
Citation: Wang, W., Pan, Q., He, F., Akhunova, A., Chao, S., Trick, H., Akhunov, E. 2018. Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat. The CRISPR Journal. 1.
DOI: https://doi.org/10.1089/crispr.2017.0010

Interpretive Summary: A gene-editing system known as CRISPR-Cas9 (CRISPR) has recently become a powerful tool for conducting strategic modifications of genes of interest. Certain variations of the CRISPR system allow multiple genes to be targeted for modification simultaneously. Here, we applied CRISPR to wheat to target genes associated with grain size and disease resistance. CRISPR-based editing of a set of three genes governing seed size led to a wheat plant that yielded plants with larger seed and increased grain weight, indicating that CRISPR can be used to increase yield in wheat. In addition, this research showed that the CRISPR system remains active in the plant through multiple generations, thereby making it possible to recover edits in later generations that may not have occurred in the initial generation. In other words, CRISPR activity is transgenerational and can facilitate the recovery of edits across multiple gene targets. Targeted gene edits generated in a given wheat line may be transferred to other wheat lines through conventional crossing allowing edited genes to be widely exploited in breeding programs for wheat improvement.

Technical Abstract: 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 rRNA-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.