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. 2020 Feb 29;48(6):2841–2852. doi: 10.1093/nar/gkaa124

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© The Author(s) 2020. 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 Non-Commercial 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 3. — GO is highly modular and can be functionally exploited to enrich base editing activity. (A) (top) Schematic of an all-in-one mScarlet-I GO reporter with (i) modified 5′ end in the mScarlet-I cDNA to include the silent start site ‘ACG’ within the 20bp sgGO complementary sequence, (ii) sgGO and (iii) neomycin selection gene (together, Scar^GO). (bottom, left) Fluorescence microscopy of 231s expressing no editor (WT) or FNLS 3 days after infection with Scar^GO. (bottom, right) Flow cytometry of 231s expressing no editor (WT) or FNLS 3- and 8-days after infection with Scar^GO. (B) (top) Schematic of an all-in-one-Luciferase GO reporter with (i) a modified 5′ end in the Luciferase2 cDNA to include the silent start site ‘ACG’ within the 20 bp sgGO complementary sequence and (ii) sgGO (together, Luc2^GO). (bottom) Luminescence in 231s and immortalized MEFs expressing no editor (WT) or FNLS 3 days after infection with and without Luc2^GO. (C) (top) Schematic of an all-in-one Blasticidin GO reporter with (i) modified 5′ end in the Blasticidin (Blas) resistance gene to include silent start site ‘ACG’ within the 20-bp sgGO complementary sequence (ii) sgGO and (iii) an endogenous targeting sgRNA under an independent U6 promoter (together, Blas^GO). (bottom) GEMSA staining/colony forming assay of Blas-treated DLD1 cells with FNLS or no editor (WT) infected with Blas^GO containing sgRNAs targeting EMX1 or no sgRNA. (D) Deep sequencing analysis of corresponding loci quantifying editing events as a fraction of total reads in DLD1 cells with Blas^GO (containing gRNAs EMX1.259, FANCF.S1, CTNNB1.S33) (left) transduced or (right) transfected with and without FNLS or Blas treatment. (E) Quantification of editing events for two off-target sites corresponding to two endogenous gRNAs EMX1.259, FANCF.S within Blas^GO in DLD1 cells (left) transduced or (right) transfected with FNLS (F) Ratio of on target to off target editing events for the OT sites with editing >1% in DLD1 cells transduced or transfected with FNLS. (G) Brightfield images of murine pancreatic organoids with inducible base editor enzyme infected with Blas^GO and treated with and without Blas. (H) Sanger sequencing chromatograms of CR8 locus in pancreatic organoids expressing BE enzyme and infected with Blas^GO (containing no gRNA or sgRNA targeting CR8) with and without Blas treatment. (I) Sanger sequencing chromatograms of Apc.1529 locus in pancreatic organoids expressing BE enzyme and infected with Blas^GO (containing no gRNA or sgRNA targeting Apc.1529) with and without Blas treatment.