Fig 2: In vitro screening for highly effective and specific gRNAs for target genes.a The target locations of gRNA in Aqp1, Adrb2, Rock1and Rock2. b CasRx expression vectors (Vector I) with GFP fluorescence signal and gRNA expression vectors (Vector II) with mCherry separate targeting Aqp1, Adrb2, Rock1and Rock2 were constructed. c Knockdown efficiency of five different gRNAs targeting the Rock1 gene in N2a cells, n = 3. Compared with group CasRx, the gRNA4 showed the highest knockdown efficiency, P = 0.0001. d Knockdown efficiency of six different gRNAs targeting the Rock2 gene, n = 3. Compared with group CasRx, the gRNA4 showed the highest knockdown efficiency in N2a cells, P < 0.0001. e The knockdown efficiency of six different gRNAs targeting Aqp1 gene was verified in stable transfected N2a monoclonal cell strain No. 6 with high expression of Aqp1 gene, n = 3. Compared with group CasRx, the gRNA3 showed the most potent knockdown efficiency, P < 0.0001. f Knockdown efficiency of four different gRNAs targeting Adrb2 gene in No. 16 stable N2a cell lines with high expression of the target gene, n = 3. Compared with group CasRx, the gRNA3 showed the strongest knockdown potential, P = 0.0032. All values were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns no signafance, two-tailed, unpaired T-test. Source data are provided as a Source Data file.

Fig 3: CasRx disrupted the target gene transcript in mice.a Experimental strategy of magnetic beads-induced and dexamethasone acetate induced intraocular hypertension mouse modes. b Three kinds of AAV virus vector encoding CasRx and gRNAs with the highest knockout efficiency. c–e Compared with non-target LacZ virus (n = 5), the CasRx system delivered by shH10 Y25 (AAV-EFS-CasRx/U6-Rock1-Rock2) virus (n = 6), significantly disrupted the expression levels of Rock1 and Rock2 genes in the TM of the mouse model injected with microbeads (P = 0.0020, P = 0.0069). The figure (d, e) were the statistical results of Rock1 and Rock2 expression levels after semi-quantification by immunofluorescence. Rock1/ Rock2 saline, n = 5; Rock1 WT, n = 5; Rock2 WT, n = 6. f–h The delivery of CasRx system by shH10 Y25 into the eye can significantly reduce the expression levels of Rock1 and Rock2 genes in trabecular meshwork of DEX-induced mice (P = 0.0147, P = 0.0218). The g and h images were fluorescence quantization results. Rock1 WT /saline/LacZ/Y25, n = 5, 6, 5, 6; Rock2 WT /saline/LacZ/Y25, n = 6, 6, 7, 5. i–k In contrast to non-target LacZ virus, shH10 Y26 (AAV-EFs-CasRx/U6-Aqp1-Adrb2) virus delivery of CasRx system into the eye can significantly damage Aqp1 and Adrb2 gene transcripts in the ciliary body of magnetic bead model mice (P = 0.0192, P = 0.0121). The expression levels of Aqp1 and Adrb2 were determined by semi-quantitative immunofluorescence analysis in (i) and (j) charts. Adrb2 Y26, n = 6; other group, n = 5. l–n shH10 Y26 (AAV-EFs-CasRx/U6-Aqp1-Adrb2) virus delivery of CasRx system into the eye can significantly interfere with Aqp1 and Adrb2 gene expression in the ciliary body of DEX-induced model mice (P = 0.0101, P = 0.0013). The expression levels of Aqp1 and Adrb2 in m-chart and n-chart were determined by semi-quantitative immunofluorescence analysis. Aqp1 WT /saline/LacZ/Y26, n = 8, 6, 5, 6; Adrb2 WT /saline/LacZ/Y26, n = 6, 7, 6, 6.The box shows TM. In all figures, scale bars, 100 μm. All values were expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance. two-tailed, unpaired T-test; Source data are provided as a Source Data file.

Fig 4: Chemerin promotes CMKLR1-RhoA/ROCK and EMT in SK-OV-3 cells. (a) The phase contrast image of SK-OV-3 cells after 24 h treatment with chemerin (50 ng/mL). Scale bar = 50 μm. (b) Western blot analysis of CMKLR1, RhoA, ROCK1, and MLC protein expressions and the level of p-MLC in SK-OV-3 and OVCAR-3 cells treated with chemerin (50 ng/mL) for 24 h. β-actin is used as a loading control. (c) Relative quantification of CMKLR1, RhoA, and ROCK1 expressions to β-actin expression, and the phosphorylation level MLC relative to MLC in B. (d) The mRNA levels of genes involving Cmklr1, Krt7, 14, 19, E-cadherin, N-cadherin, vimentin, and Zeb1. β-actin and GAPDH were used as internal reference genes. (e) Western blot analysis of N-cadherin, E-cadherin, vimentin, and ZEB1 expressions in SK-OV-3 cells treated with chemerin (50 ng/mL) for 24 h. β-actin is used as a loading control. (f) Quantification of N-cadherin, E-cadherin, vimentin, and ZEB1 expressions in E. ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. Data are plotted as the mean ± SD.

Fig 5: The silencing of CMKLR1 reverses chemerin-enhanced invasion of SK-OV-3 cells and RhoA/ROCK/EMT activation. (a) Western-blot analysis of CMKLR1, RhoA, ROCK1, MLC, p-MLC, N-cadherin, E-cadherin, and vimentin in SK-OV-3 cells silenced with siRNAs targeted with CMKLR1. (b) Relative quantification of CMKLR1, RhoA, and ROCK1 expressions to β-actin and the phosphorylation level of MLC relative to MLC in A. (c) Quantification of N-cadherin, E-cadherin, and vimentin expressions relative to β-actin expression in A. (d) The invasion of SK-OV-3 cells silenced with CMKLR1 siRNA was measured using transwell assay. (e) The invasion of SK-OV-3 and OVCAR-3 cells silenced with siRNAs targeted with CMKLR1 was measured using transwell. Scale bar = 100 μm. (f) Quantification of invading cell numbers in the transwell experiment. Significance was determined by one-way ANOVA analysis with Tukey's post hoc test. ∗∗∗∗,####P < 0.0001. Data are plotted as the mean ± SD.