Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat

Abstract

BackgroundRecently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium-mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation.ResultsIn the present study, we efficiently applied targeted mutagenesis in common wheat (Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Seven target sites in three genes (Pinb, waxy and DA1) were selected to construct individual expression vectors. The activities of the sgRNAs were evaluated by transforming the constructed vectors into wheat protoplasts. Mutations in the targets were detected by Illumina sequencing. Genome editing, including insertions or deletions at the target sites, was found in the wheat protoplast cells. The highest mutation efficiency was 6.8% in the DA1 gene. The CRISPR/Cas9 binary vector targeting the DA1 gene was then transformed into common wheat plants by Agrobacterium tumefaciens-mediated transformation, resulting in efficient target gene editing in the T0 generation. Thirteen mutant lines were generated, and the mutation efficiency was 54.17%. Mutations were found in the A and B genomes of the transgenic plants but not in the D genome. In addition, off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences.ConclusionsOur results showed that our Agrobacterium-mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1496-x) contains supplementary material, which is available to authorized users.

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

“…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.…”

“…In addition to TaCKX2‐1 , we also obtained mutation in three other grain regulatory genes, TaGLW7 , TaGW2 , and TaGW8 . Two recent studies on TaGW2 showed that knockout mutations of this gene could significantly increase wheat grain size and grain weight (Wang et al ., ; Zhang et al ., ,). Combining and transferring the mutations of TaCKX2‐1 and TaGW2 into the elite wheat cultivars are expected to have an additive effect on increasing the sink strength.…”

“…These improvements have not been integrated into CRISPR/Cas9-based genome editing or other genome editing platforms in wheat. During the preparation of this manuscript, a report was published on Agrobacterium-delivered CRISPR/Cas9 for wheat gene editing in which one gene (DA1) was tested in T 0 transgenic plants with a high frequency of not yet proven inheritable mutations (Zhang et al, 2018a). In the present research, we developed an Agrobacterium-delivered CRISPR/ Cas9 system for genome editing in wheat and applied it to target four grain-size regulatory candidate genes.…”

TaDA1, a conserved negative regulator of kernel size, has an additive effect with TaGW2 in common wheat (Triticum aestivum L.)

“…Recently, gene editing has been widely applied to improve the agronomic traits of crops (Wang et al , ; Zhang et al , ). It is possible that this technique can be used to knock out DA1 and its close homologs (Zhang et al , ), potentially generating a much greater impact on wheat kernel size. Furthermore, our proteomic data showed that TaDA1 and TaGW2 affected partially overlapping but relatively independent protein networks involved in diverse pathways (Figure a, b), including protein and starch biosynthesis, cell proliferation, and cell expansion.…”