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. 2023 Mar 28;6:334. doi: 10.1038/s42003-023-04708-2

Fig. 1. Targeting a DsRed reporter mRNA with CRISPR/RfxCas13d causes collateral activity in human cells.

Fig. 1

HEK293T cells were transfected with DsRed, BFP, RfxCas13d-2A-GFP, and either a control gRNA or a DsRed-targeting gRNA. (a) representative fluorescence imaging data. (b) quantification of (a) (N = 4). (c) qRT-PCR (N = 3). (d) Western blot with anti-HA. (e), scatter plot of DsRed and GFP mRNA levels measured by qRT-PCR for ten gRNAs targeting DsRed. Red dot indicates the gRNA used throughout this study. (f) total RNA quantified by NanoDrop (N = 3). g, h poly(A) RNA-seq (g N = 3, h N = 2, replicates were pooled). For qPCR and RNA-seq, 5% mouse MEF-1 cells were spiked into equal number of HEK293T cells prior to RNA extraction. Mouse Gapdh (mGapdh) was used as load control in (c). 5,000-fold less DsRed plasmid was used in (h). (i) BFP signal when a 2-fold dilution series of DsRed was used (quantified in j N = 3). Two-tailed Student’s t-test was used for (b, c, f). **p < 0.01. l.e. long exposure. Error bars represent standard deviation.