BioDesign Research / 2021 / Article / Fig 2

Research Article

Durable CRISPR-Based Epigenetic Silencing

Figure 2

Assay for assessing long-term gene silencing. (a) A fluorescent GFP reporter driven by the SV40 promoter is integrated into HEK293T cells, along with a cassette that guides dCas9 to bind to the SV40 promoter. These cells can be transiently transfected with dCas9 fused to various domains to assess the ability to repress GFP over time. (b, c) GFP expression over time following transfection of effectors. Dots indicate individual time points, and model fit is indicated by solid lines. (b) Transfection of dCas9-KRAB. (c) Cotransfection of dCas9-KRAB, dCas9-DNMT3A, and dCas9-DNMT3L. (d) Long-term silencing ability of various effectors individually (gray) or in combinations of two domains (blue) or three (yellow) domains. Dashed lines indicate predicted value if the relevant domains’ silencing activity adds independently.