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. 2017 Apr 1;28(7):898–906. doi: 10.1091/mbc.E17-01-0051

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© 2017 Katoh et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Schematic overview of CRISPR/Cas9-mediated knock-in system. (A) RPE1 cells are transfected with an all-in-one vector, which contains spCas9 and an sgRNA expression cassette, together with a donor vector (pDonor-tBFP-NLS-Neo), which contains tBFP-3×NLS and Neo as selection markers. The RNA-guided Cas9 concurrently cleaves target sequences in the genome and donor vector. The DSBs trigger HIDR, in which the linearized donor vector can be integrated into the target locus in a forward or reverse direction. One of three types of donor integration can occur: (a) no integration of the donor vector, but with small indels; (b) forward integration of the donor vector; or (c) reverse integration of the donor vector. (B) Merged image of differential interference contrast and fluorescence microscopy. Signals of nuclear tBFP suggest successful knock-in of the donor vector. Scale bar, 20 μm.