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. 2018 May 24;9:699. doi: 10.3389/fpls.2018.00699

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Copyright © 2018 Liang, Han, Wang, Jiang and Xu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The USTA gene and deletion mutants. (A) The USTA gene and gRNA spacers. The position and direction of gRNA spacers and primers used to generate and screen ustA deletion mutants are marked with arrows. The on-target scores of spacers UA01, UA13, and UA21 are labeled in the bracket. (B) PCR assays for the deletion of USTA (upper panel) and presence of the geneticin-resistance gene (lower panel) in 20 transformants generated with pCas9-tRp-gRNA-UA01 (spacer UA01). The 470-bp USTA fragment was only amplified in the wild-type (CK) and transformant 18. M: 1-kb DNA ladder marker. (C) PCR assays to verify gene replacement events in 20 putative ustA deletion mutants. The 1576-bp upstream and 1686-bp downstream recombination products were amplified with primer pairs USTA7F(U7F)/G855R and USTA8R(U8R)/G856F, respectively. Amplification with P1 was used as the negative control. M: 1-kb DNA ladder marker.