Fig. 2
From: Cas13b-mediated RNA targeted therapy alleviates genetic dilated cardiomyopathy in mice
Specific knockdown of hTNNT2R141W in vitro using PspCas13b. (A) Schematic showing the experimental procedure for the delivery of PspCas13b and sgTNNT2-expressing AAV9s into hTNNT2R141W transgenic mice. (B) Schematic representation of the mechanism of the PspCas13b-sgRNA system in silencing the expression of the mutant TNNT2R141W transcript. (C) qRT-PCR demonstrating successful knockdown of hTNNT2R141W in TNNT2R141W-293T cells (n = 4 per group). ***p < 0.001 according to Student’s t test. (D) Schematic diagram of the process for constructing an TNNT2WT/R141W-AC16 cell line using SpRY-CBEmax. (E) Schematic representation of the process of PspCas13b-specific knockdown of mutant transcript expression in the TNNT2WT/R141W-AC16 cell line and the HEK-293T cell line with reporter alleles. (F) Assessment of knockdown of WT (green) and mutant (red) mRNA expression in HEK-293T cells transfected with reporter alleles and PspCas13b-sgRNA2 normalized to the levels observed in cells transfected only with the reporter alleles (n = 3 independent experiments). (G) qRT-PCR quantification of total TNNT2 mRNA in TNNT2WT/R141W-AC16 cells (n = 6 independent experiments). *p < 0.05 according to Student’s t test. (H) Sanger sequencing of T vectors with WT and hTNNT2R141W transcripts. (I) Expression levels in log2 (fragments per kilo base per million mapped reads [FPKM] + 1) values of all detected genes in RNA sequencing (RNA-seq) libraries of PspCas13b-TNNT2 (y axis) compared to PspCas13b control (x axis) (n = 3 independent replicates for both groups)