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

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 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.

Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

“…We were not able to detect a mutation in T 0 plants by T7E1 assay and Sanger sequencing directly on PCR products. This result could be due to low edit frequency in wheat as shown in prior studies (Wang et al ., , ), but somatic mutations may have occurred in the T 0 plants. To test this hypothesis, we sequenced the PCR products derived from the TaCKX2‐1 target sites by the HiSeq 2000 platform.…”

“…CRISPR/Cas9 has been the primary choice for plant genome editing, and Agrobacterium ‐mediated transformation is the most common method for delivery of the CRISPR/Cas9 DNA components to plants (Soyars et al ., ). Mainly due to historical reason, all genome editing systems in wheat were implemented through the biolistics‐mediated transformation (Liang et al ., ; Sánchez‐León et al ., ; Wang et al ., , ; Zhang et al ., ,). Co‐transfer of large DNA fragments from the vector backbone by biolistic transformation leads to the high frequency of transgene silencing (Anand et al ., ; Tassy et al ., ).…”

“…In T 0 plants, deep sequencing indicates that over 99% of the mutations are due to deletions, and our genotyping data from T 1 and T 2 plants show that only five insertions were found of the 68 mutations detected, all of which are insertions of 1‐bp A or T. Remaining 63 mutations are deletions (Figure S11). Similar scenarios were also found in other wheat studies (Liang et al ., ; Sánchez‐León et al ., ; Wang et al ., , ; Zhang et al ., ,). Thus, deletions are the dominant type of edit mutations in wheat.…”

“…Therefore, the potential edit rate in TaCKX2‐1 could be even higher. This is approximately threefold higher than the previously reports in wheat by Biolistic delivery (Liang et al ., ; Sánchez‐León et al ., ; Wang et al ., , ). Compared to TaCKX2‐1 and TaGW2 , the mutation rate in TaGLW7 and TaGW8 is much lower.…”

“…In this respect, the CRISPR technology is expected to contribute greatly to creating novel variations in wheat. Wheat was one of the plant species first modified by CRISPR/Cas9 (Shan et al ., ), and several traits have been targeted for mutations, including powdery mildew resistance (Wang et al ., ), grain size (Wang et al ., ; Zhang et al ., ), and grain quality (Liang et al ., ; Sánchez‐León et al ., ; Zhang et al ., ). In all these researches, the Cas9 and sgRNA transgenes were delivered by the biolistic bombardment.…”