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. 2016 Jul 25;44(19):e149. doi: 10.1093/nar/gkw660

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Inducible and multiplexed gene knockout in vitro and in vivo. (A) NotI and XhoI digestion of indicated plasmids showing correct vector assembly of 2, 3, 4, 5 or 6 sgRNA cassettes. LG, LentiGuide. M, Log-2 DNA ladder (NEB). Fragments carrying sgRNA cassettes were indicated by stars (*). (B) A summary of the cloning efficiency for making the correct constructs with different numbers of sgRNAs. Ten clones were randomly picked and verified by the size of NotI and XhoI digested fragments. (C) HeLa/iCas9-c1 cells were transduced with LentiGuide or Lenti-entry-puro carrying the indicated sgRNA(s). Cells were then treated with or without 1 μg/ml DOX for 48 h and harvested for western blot analyses. (D) Western blot analyses of tumor tissues isolated from the mice injected with indicated cells and fed with control or DOX chow.