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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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Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of BsaI-digested pUC19-tRp-gRNA (upper) or BsmBI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme BsaI and BsmBI are marked with black arrows.