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. 2020 Jun 10;11:769. doi: 10.3389/fpls.2020.00769

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Copyright © 2020 Brandt, Gunn, Moretti and Zemetra.

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(s) 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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Overview of the protoplast isolation and transformation protocol. Leaf tissue is cut into 2 mm pieces and digested in an enzymatic solution on a shaker. The protoplasts are then filtered and isolated using a sucrose gradient. A PEG-mediated transformation is performed in microcentrifuge tubes, using GFP as a positive control. After 24–48 h, the transformation efficiency is assessed by counting GFP fluorescing cells in the positive control. DNA is isolated from CRISPR-edited protoplasts and PCR is performed. The PCR is then used in a T7EI digestion assay as well as Sanger sequenced and analyzed using an online program to assess the editing efficiency of the chosen sgRNA.