Extended Data Fig. 4: Screening of candidate pre-gRNAs targeting mouse Ctsl in vitro and examination of the editing specificity of LNP-CasRx-pre-gCtsl in vivo.
From: Cas13d knockdown of lung protease Ctsl prevents and treats SARS-CoV-2 infection
a, Schematic of CasRx and pre-gRNA plasmid system for screening candidate pre-gRNA targeting mouse Ctsl. Relative Ctsl mRNA expression was measured by RT-PCR after 48 h transfection with b, Plasmids encoding CasRx with different pre-gRNAs designed for targeting Ctsl; or c, CasRx mRNA and selected pre-gCtsl from (b) in mouse KLN205 cells. n = 4 biologically independent samples for b; n = 5 biologically independent samples for c. Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t-tests. ****P < 0.0001. d, Kinetics of CasRx mRNA in lung. C57BL/6 mice were administered LNPs encapsulating CasRx mRNA at 0.5 mg/kg by tail vein. Lungs were collected at 0, 4, 14, 24 or 48 h, respectively, after the injection for qRT-PCR analysis. Data are presented as mean ± SEM for each time point (n = 4 for 0, 48 h; n = 5 for 4 h, 14 h, 24 h). e-f, C57BL/6 mice were administered DPBS, eLNPs, LNP-CasRx-pre-gControl or LNP-CasRx-pre-gCtsl by tail vein. After 72 h, the lung, liver and spleen were collected for analysis. e, Ctsl mRNA levels in liver and spleen remained unchanged by LNP-CasRx-pre-gCtsl treatment. f, mRNA levels of isoform Ctsd, Ctsb and Ctss in lung are unchanged by LNP-CasRx-pre-gCtsl treatment. Transcript levels were normalized to Gapdh and presented as mean ± SEM. n = 4 per group, 2 females and 2 males. P values were calculated by one-tailed Mann-Whitney U test. NS, not significant.
