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. 2016 Feb 4;44(9):e85. doi: 10.1093/nar/gkw064

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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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — Comparison between HDR- and NHEJ-mediated reporter knock-in. (A) Schematics showing a zoomed-in view of the sg-1–4 target sites and their positions on the genomic GAPDH locus, as well as the design of ires-eGFP(+HAs) Donor-1, 2, 2.A and 2.B plasmids. Homology arm regions used in the ires-eGFP(+HAs) donor-1 are highlighted in gray, and the HAs used in donor-2, 2.A and 2.B are highlighted in purple. Donor-2.A carries a single sg-A target site at the 3′, and Donor-2.B carries a sg-A target site at the 5′ of the ires-eGFP(+HAs) cassette. (B) FACS analysis of LO2 cells transfected with the ires-eGFP(+HAs) donor-1/Cas9 and sg-1, 2, 3 or 4. Due to the different target positions on the genome and the donor, sg-1 induced HDR-mediated knock-in; sg-2 and sg-3 induced NHEJ-based knock-in; and sg-4 mainly produced GFP+ cells via the HDR-based knock-in through the intact 5′ homology arm. (C) FACS analysis showing HDR-mediated knock-in with circular and linear donor templates. The ires-eGFP(+HAs) Donor-2, 2.A or 2.B were transfected together with Cas9/sg-1 or Cas9/sg-2. The Donor-2.A and 2.B were both examined in the presence of sg-A (linear) as well as in the absence of sg-A (circular). Cas9/sg-A cleaves the Donor-2.A at 3′ of the ires-eGFP(+HAs) cassette and the linearized Donor 2.A produced GFP+ cells via HDR-mediated knock-in. Distinctly, Cas9/sg-A cleaves the Donor-2.B at 5′ of the ires-eGFP(+HAs) cassette, and the linearized Donor 2.B produced high proportion of GFP+ cells via both NHEJ- and HDR-mediated knock-in. (D) FACS results showing NHEJ- and HDR-mediated reporter knock-in at ACTB, SOX17 and T gene loci. Upper panel shows the schematics of ires-eGFP and PGK-eGFP reporters used for knock-in at ACTB and SOX17 or T gene loci, respectively. Single-cut NH-donor was co-transfected with Cas9/sg-A/sgACTB-i or sgACTB-ii to target the ACTB locus (lower left panel, top two rows); while the CE NH-donor was co-transfected with Cas9/sg-A/sgSOX17-i, sgSOX17-ii or sgT-i to target the SOX17 or T gene loci (lower right panel, top two rows). ACTB HDR-donor carrying ires-eGFP, and SOX17 and T HDR-donors containing PGK-eGFP, were co-transfected with Cas9 and corresponding sgRNAs to examine the HDR-based knock-in (lower panel, bottom row). Control samples were transfected without gene-specific sgRNA or sg-A. FACS analysis for the tests at ACTB locus was performed at day 5 after transfection. Cells transfected with PGK-eGFP containing donors for the tests at SOX17 and T loci, were maintained for five passages before FACS analysis. GFP+ cells are gated to the right of the dashed line in each panel.