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. Author manuscript; available in PMC: 2019 Sep 26.
Published in final edited form as: J Am Chem Soc. 2018 Sep 18;140(38):11974–11981. doi: 10.1021/jacs.8b05012

Figure 2.

Figure 2.

dCas13b-YTHDF fusion proteins can trigger enhanced protein production or RNA knockdown on a reporter transcript. (A) Vector system used to test targeted reader protein tools in a dual luciferase assay. Firefly luciferase (Fluc) is the target of the experiment, while Renilla luciferase (Rluc) is an internal control. RNA levels are monitored by RT-qPCR, and protein production is monitored by luciferase luminescence. Anoff-target gRNA (NT) and a guide RNA targeting Fluc (Fluc) delivery the fusion protein to the transcript. (B) HEK293T cells were co-transfected with the vectors shown in (A), including the active Cas13b nuclease, and 48 h after transfection subjected to RT-qPCR (red bar) and luciferase assay (blue bar). (C) HEK293T cells were co-transfected with the vectors shown in (A), with the dCas13b-YTHDF1 fusion, and assayed by RTqPCR (red bar) and luciferase assay (blue bar). (D) HEK293T cells were co-transfected with the vectors shown in (A), with the dCas13b-YTHDF2 fusion, and assayed by RT-qPCR (red bar) and luciferase assay (blue bar). Student’s t-test; *P < 0.05, **P < 0.01 (n = 3 for RTqPCR, n = 3 biological × 2 technical for Cas13b and dCas13b-Y2 luciferase assays, n = 3 biological × 2 technical + 3 biological replicates for dCas13b-Y1 luciferase assay).