Integrated gene-free potato genome editing using transient transcription activator-like effector nucleases and regeneration-promoting gene expression by Agrobacterium infection

Abstract

Genome editing is highly useful for crop improvement. The method of expressing genome-editing enzymes using a transient expression system in Agrobacterium, called agrobacterial mutagenesis, is a shortcut used in genome-editing technology to improve elite varieties of vegetatively propagated crops, including potato. However, with this method, edited individuals cannot be selected. The transient expression of regeneration-promoting genes can result in shoot regeneration from plantlets, while the constitutive expression of most regeneration-promoting genes does not result in normally regenerated shoots. Here, we report that we could obtain genome-edited potatoes by positive selection. These regenerated shoots were obtained via a method that combined a regeneration-promoting gene with the transient expression of a genome-editing enzyme gene. Moreover, we confirmed that the genome-edited potatoes obtained using this method did not contain the sequence of the binary vector used in Agrobacterium. Our data have been submitted to the Japanese regulatory authority, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and we are in the process of conducting field tests for further research on these potatoes. Our work presents a powerful method for regarding regeneration and acquisition of genome-edited crops through transient expression of regeneration-promoting gene.

Keywords: TALEN; genome editing; regeneration-promoting gene; steroidal glycoalkaloids; transient gene expression.

Figure 1. Completely SSR2-disrupted lines from “Sayaka”. (A) Sequence of target site of SSR2 gene was amplified, cloned to E. coli, and sequenced. Pale blue indicates sequence targeted by TALEN. (B) SGA contents of in vitro shoots. Error bars indicate the standard deviation of three technical replicates. (C) Tubers harvested in green house under artificial light.

Figure 2. Verification of the absence of vector sequence using PCR. NT; non-transgenic “Sayaka”, No.; PCR set number in Supplementary Table S2. The bands that amplify in columns containing vector DNA are the sequences amplified by the PCR method. PCR18 amplifies fragments of two sizes. PCR19, PCR20 amplify fragments of three or more sizes and are representative of some of them.

Figure 3. Detection of identical k-mers between the genome-edited potato genome and vector sequences. Data obtained from the wild-type (WT)(A) and selected genome-edited potato lines #117 (B), and #164 (C) are shown for k=20 (left) and 25 (right). The results of the counts and G-statistic values (against wild-type) at each position in the pSuehiro108 vector sequence used in genome-editing are shown in vertical plots. The red horizontal line corresponds to the 1% significance level (G-values greater than 6.634) and the vertical plot exceeding this line are shown in red. The k-mer analysis results obtained by repeated NGS experiments are summarized in Supplementary Tables S3, S4.

Figure 4. Schematic explanation for positive selection using transient expression of regeneration-promoting (Reg) gene.