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. Author manuscript; available in PMC: 2023 Jan 3.
Published in final edited form as: Cell Rep. 2022 Aug 16;40(7):111226. doi: 10.1016/j.celrep.2022.111226

Figure 3. Targeting endogenous mRNAs with Cas13d activates collateral RNase activity.

Figure 3.

(A) Description of collateral activity RNA assay. HeLa-tet:Cas13d-mCherry cells express mCherry constitutively and Cas13d under a doxycycline-inducible promoter. As collateral activity likely depletes transgenic and endogenous RNAs, non-transgenic HeLa cells are spiked in prior to transfection of gRNA and induction of Cas13d. After 44 h, mCherry and GAPDH mRNA are measured by qRT-PCR.

(B) Assay validation using CUG-targeting gRNAs and transfected CUG480. n = 3 transfections, averaged across two qPCR replicates. Error bars show SEM of ΔCq propagated to the plotted ratio. *p < 0.05, **p < 0.01, ***p < 0.001, one-tailed t test of ΔCq.

(C) Ratio of mCherry to GAPDH mRNA when targeting endogenous genes. Target expression was calculated from RNA-seq of untransfected HeLa cells included in our experiment described in Figure S2. n = 3 transfections, averaged across two qPCR replicates. Error bars show SEM of ΔCq propagated to the plotted ratio. Gray line depicts a logistic function fit using non-linear least-squares regression. *p < 0.05, one-tailed t test of ΔCq.