Peculiar properties of tuber starch in a potato mutant lacking the α-glucan water dikinase 1 gene <i>GWD1</i> created by targeted mutagenesis using the CRISPR/dMac3-Cas9 system

Abstract

Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1 -deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze–thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1 , sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.

Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy

“…This is because the use of highly efficient parental lines allows maintaining a higher level of Cas9 activity ( Miki et al., 2018 ; Zhang et al., 2022 ). Transcriptional and translational enhancers have been applied for mutagenesis purposes ( Gasparis et al., 2018 ; Kusano et al., 2018 ; Onodera et al., 2018 ; Takeuchi et al., 2021 ; Kusano et al., 2023 ; Ohnuma et al., 2023 ), but rarely for HDR-mediated GT ( Peng et al., 2020 ). During the preparation of this manuscript, it has been reported that the intron-containing version of ttCas12a showed higher GT efficiency than the unmodified ttCas12a ( Schindele et al., 2023 ).…”

“…In this study, a small but significant differences were detected in the enhancer combination, but drastic improvements must be obtained if a version without enhancers is used as a control. Based on previous reports ( Gasparis et al., 2018 ; Kusano et al., 2018 ; Onodera et al., 2018 ; Takeuchi et al., 2021 ; Kusano et al., 2023 ; Ohnuma et al., 2023 ), the AtUbq10 first intron and dMac3 is presumed to be an ideal option for improving efficiency, but further analysis is needed, including analysis of individual enhancers separately, as it is also speculated that the combination of enhancers may have some negative effects.…”

“…Using the first intron of Arabidopsis Ubiquitin 10 (AtUbq10) as a transcriptional enhancer, Cas9-mediated heritable mutants were generated at high frequency in barley ( Gasparis et al., 2018 ). Furthermore, dMac3, a highly efficient translational enhancer of the rice OsMac3 gene, increased the efficiency of targeted mutagenesis by Cas9 and TALEN in rice and potato ( Kusano et al., 2018 ; Onodera et al., 2018 ; Takeuchi et al., 2021 ; Kusano et al., 2023 ; Ohnuma et al., 2023 ). These enhancers have not been applied much to mutagenesis or GT with the CRISPR/Cas systems, and combinations of enhancers have not yet been reported.…”