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

FIGURE 1:

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.