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. 2014 Oct 15;4(12):2409–2418. doi: 10.1534/g3.114.013979

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Copyright © 2014 Zhang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Cloning scheme and sgRNA activity tests. (A) Single-step Golden Gate assembly scheme of the STOP-dsRed donor vector cloned into a modified pBluescript backbone. (B) Scheme of the “generic” attB plasmids used in our study. A simple cloning step is sufficient to generate any gene-specific attB plasmid that can be used to replace an excised exon. (C) sgRNA synthesis scheme. (D) sgRNA activity assay of 12 different sgRNAs in S2 cells. PCR result with (bottom) or without (middle) T7-Endonuclease I treatment are shown. Digested products are marked by arrow heads and effective sgRNAs are marked by a red asterisk. C are controls without sgRNA.