Fig. 1.

Screening for efficient and specific sgRNAs for targeting the Tmc1^Bth transcript. a Constructs used for the sgRNA screen mediated by the Cas RNA editing system. Five vectors were constructed, including the Cas expression vector, the mCherry-Tmc1^Bth fluorescence reporter, the mCherry-Tmc1⁺ fluorescence reporter, the sgRNA expression vectors for targeting the Tmc1^Bth transcript, and the non-targeting (NT) sgRNA expression vector. b Ratios of fluorescence intensity with sgRNAs compared to control sgRNA (NT) for targeting mCherry-Tmc1^Bth mRNA and mCherry-Tmc1⁺ mRNA mediated by CasRx system. Data are shown as the mean ± SD (n = 3 biologically independent samples). c Ratios of fluorescence intensity with sgRNAs compared to control sgRNA (NT) for targeting mCherry-Tmc1^Bth mRNA and mCherry-Tmc1⁺ mRNA mediated by PspCas13b system. Data are shown as the mean ± SD (n = 3 biologically independent samples). d Mean ratio of fluorescence intensities between mCherry-Tmc1^Bth and mCherry-Tmc1⁺ mRNA. sgRNA3 has the lowest mean ratio of fluorescence intensity for all the 30 sgRNAs tested. e The integrated fluorescence intensity of cells with CasRx system. The integrated fluorescence density was significantly decreased with sgRNA3 targeting mCherry-Tmc1^Bth mRNA compared to targeting mCherry-Tmc1⁺ mRNA, and integrated fluorescence density was decreased comparted to targeting mCherry-Tmc1^Bth and mCherry-Tmc1⁺ mRNA with non-targeting sgRNA. Data are shown as the mean ± SD (n = 5 biologically independent samples). ***p < 0.001, P-values were determined by one-way ANOVA with Sidak’s multiple comparisons test