Lin XL et al. (2025), CRISPR/Cas-mediated mRNA knockdown in the embry...

XB-ART-61356

Cell Biosci 2025 Apr 23;151:52. doi: 10.1186/s13578-025-01397-8.

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CRISPR/Cas-mediated mRNA knockdown in the embryos of Xenopus tropicalis.

Lin XL , Zhou YM , Meng K , Yang JY , Zhang H , Lin JH , Wu HY , Wang XY , Zhao H , Feng SS , Park KS , Cai DQ , Zheng L , Qi XF .

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The Xenopus tropicalis (Western clawed frog) is an important amphibian model for genetics, developmental and regenerative biology, due to its diploid genetic background and short generation time. CRISPR-Cas13 and CRISPR interference (CRISPRi) systems have recently been employed to suppress mRNA expression in many organisms such as yeast, plants, and mammalian cells. However, no systematic study of these two systems has been carried out in Xenopus tropicalis. Here, we show that CRISPRi rather than CRISPR-Cas13 is an effective and suitable approach to suppress specific mRNA transcription in Xenopus tropicalis embryos. We demonstrated that CRISPRi composed of dCas9 and KRAB-MeCP2 (dCas9-KM) can efficiently target exogenous and endogenous transcripts in Xenopus tropicalis embryos. Moreover, our data suggest that the new KRAB domain from ZIM3 protein (ZIM3-KRAB, ZIM3K) alone has a comparable transcript targeting capacity in Xenopus tropicalis embryos to the traditional fusion repressor KRAB-MeCP2 in which the KRAB domain from KOX1 protein. In conclusion, our results demonstrate that CRISPRi rather than CRISPR-Cas13 is an efficient knockdown platform to explore specific gene function in Xenopus tropicalis embryos.

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Species referenced: Xenopus tropicalis
Genes referenced: tyr

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	Figure 1. Effects of PspCas13b and RfxCas13d on reporter gene expression in embryos of X. tropicalis. (A) Schematic of Cas13-related gRNAs targeting the mRNA of EGFP reporter gene. (B) Schematic illustration of the experimental setup used to analyze PspCas13b and RfxCas13d capacities to target exogenous reporter gene in X. tropicalis embryos. The mRNA of mCherry was used as the internal control. (C and D) Representative images (C) and quantification (C) of EGFP fluorescence in control and experimental embryos injected with PspCas13b system at 15 hpi (n = ~ 30 embryos from 3 independent experiments). (E and F) Representative images (E) and quantification (F) of EGFP fluorescence in control and experimental embryos injected with PspCas13b system at 24 hpi (n = ~ 30 embryos from 3 independent experiments). (G) The qPCR validation of EGFP expression in control and experimental embryos injected with PspCas13b system at 24 dpi (n = 5). (H and I) Representative images (H) and quantification (I) of EGFP fluorescence in control and experimental embryos injected with RfxCas13d system at 15 hpi (n = ~ 30 embryos from 3 independent experiments). (J and K) Representative images (J) and quantification (K) of EGFP fluorescence in control and experimental embryos injected with RfxCas13d system at 24 hpi (n = ~ 30 embryos from 3 independent experiments). (L) The qPCR validation of EGFP expression in control and experimental embryos injected with RfxCas13d system at 24 dpi (n = 5). All data are presented as mean ± SEM. Scale bar = 500 μm (C, E, and J) and 750 μm (H). Ns, no significant differences
	Figure 2. Effects of PspCas13b and RfxCas13d on tyrosinase expression in embryos of X. tropicalis. (A) Schematic of Cas13-related gRNAs targeting the mRNA of endogenous tyrosinase (tyr) gene. (B) Schematic illustration of the experimental setup used to analyze the capacities of PspCas13b and RfxCas13d systems (300 pg/embryo) together with gTyr1-3 (100 pg/embryo). (C and D) Representative images (C) and quantification (D) of Tyr expression in control and experimental embryos injected with PspCas13b system at 24 hpi. Scale bar = 500 μm. (E and F) Representative images (E) and quantification (F) of Tyr expression in control and experimental embryos injected with PspCas13b system at 48 hpi. Scale bar = 1 mm. (G and H) Representative images (G) and quantification (H) of Tyr expression in control and experimental embryos injected with RfxCas13d system at 24 hpi. Scale bar = 500 μm. (I and J) Representative images (I) and quantification (J) of Tyr expression in control and experimental embryos injected with RfxCas13d system at 48 hpi. Scale bar = 1 mm. All data are presented as mean ± SEM (n = 4 per group). Ns, no significant differences
	Figure 3. Effects of dCas9-KM system on reporter gene expression in embryos of X. tropicalis. (A) Schematic of CRISPRi-related gRNAs targeting the regions of mCherry reporter gene in pmCherry-N1 plasmid. (B) Schematic illustration of the experimental setup used to analyze dCas9-KM capacity targeting exogenous reporter gene in X. tropicalis embryos. (C) The qPCR validation of mCherry expression in embryos co-injected with dCas9-KM system and indicated gRNAs at 48 dpi (n = 4 per group). (D and E) Representative images (D) and quantification (E) of mCherry expression in embryos co-injected with dCas9-KM system and indicated gRNA at 48 hpi (n = ~ 60 embryos from 3 independent experiments). (F) Schematic of gR-187 targeting mCherry reporter gene in pCMV-mCherry-EF1α-EGFP dual-reporter plasmid. (G) The qPCR validation of mCherry expression in embryos co-injected with dCas9-KM system and indicated gRNA at 48 dpi (n = 5 per group). EGFP was used as the internal control. (H and I) Representative images (H) and quantification (I) of mCherry fluorescence in embryos co-injected with dCas9-KM system and indicate gRNA at 48 hpi (n = 24 embryos from 3 independent experiments). All data are presented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 versus control. Ns, no significant differences versus control
	Figure 4. Effects of dCas9-KM system on tyrosinase expression in embryos of X. tropicalis. (A) Schematic illustration of dCas9-KM system-related gRNAs targeting the DNA locus of endogenous tyrosinase (Tyr) gene. Fertilized eggs were co-injected with dCas9-KM mRNAs and indicated gRNA sets, followed by embryo evaluation at 48 hpi as follows. (B and C) Representative images (B) and quantification (C) of Tyr production in embryos co-injected with dCas9-KM mRNA and indicated gRNAs at 48 hpi (n = 26 ~ 30 embryos from 3 independent experiments). Scale bar = 250 μm. (D) The qPCR validation of Tyr expression in embryos co-injected with dCas9-KM mRNA and indicated gRNAs at 48 dpi (n = 5 per group). (E and F) Representative images (E, scale bar = 250 μm) and quantification (F) of embryos with different phenotypes were counted and compared with the total developed ones 48 h post co-injection of dCas9-KM system. (G and H) Representative images (G) and quantification (H) of embryos with different phenotypes were counted and compared with the total developed ones 72 h post co-injection of dCas9-KM system. Total embryos evaluated for each group (n) is shown above each column. All data are present as mean ± SEM. ****p < 0.0001 versus gNC group.
	Figure 5. Effects of dCas9-KM system on pax6 expression in embryos of X. tropicalis. (A) Schematic illustration of dCas9-KM system-related gRNAs targeting the DNA locus of endogenous pax6 gene. Fertilized eggs were co-injected with dCas9-KM mRNAs and indicated gRNA sets, followed by embryo evaluation at 48 hpi as follows. (B) The qPCR validation of pax6 expression in the dCas9-KM-injected embryos with different gRNA sets (n = 5 per group). (C) Representative images of embryos with smaller eyes in the experimental group injected with pax6 targeting gRNA sets. The top panel shows low magnification, and the lower panel shows high magnification. Asterisk denotes smaller eyes. (D) Embryos with different phenotypes were counted and compared with the total developed ones after injection. Total embryos evaluated for each group (n) is shown above each column. (E) Quantification of eye size in tadpoles treated with gRNA sets and controls (n = 3 per group). (F) The qPCR validation of pax6 expression in the dCas9-KM-injected embryos with single gRNAs from set2 (n = 5 per group). All data are present as mean ± SEM. p < 0.05, p < 0.01, **p < 0.001 versus gNC group. Ns, no significant differences versus gNC group.
	Figure 6. Dose effect of dCas9-KM system on the efficiency of Tyr knockdown in X. tropicalis embryos. (A) Representative images (left and upper panels) and qPCR validation (right and lower panel) of tyrosinase expression in embryos co-injected with gTry13 and dCas9-KM mRNAs at 48 hpi. Fertilized eggs were co-injected with gTry13 (200 pg/embryo) and dCas9-KM mRNAs with different concentrations from 0 to 600 pg/embryo. Data are presented as mean ± SEM (n = 5 per group). p < 0.01, p < 0.001, **p < 0.0001 versus control group (one-way ANOVA test). Ns, no significant differences. Scale bar = 2500 μm. (B) Representative images (left and upper panels) and qPCR validation (right and lower panel) of tyrosinase expression in embryos co-injected with gTry13 and dCas9-KM mRNAs at 48 hpi. Fertilized eggs were co-injected with dCas9-KM mRNAs (300 pg/embryo) and gTry13 with different concentrations from 0 to 600 pg/embryo. Data are presented as mean ± SEM (n = 5 per group). p < 0.05, ****p < 0.0001 versus control group (one-way ANOVA test). ####p < 0.0001 (one-way ANOVA test). Ns, no significant differences. Scale bar = 1000 μm. (C and D) Representative images (C) and quantification (D) of embryos with different phenotypes were counted and compared with the total developed ones 48 hpi. Scale bar = 750 μm
	Figure 7. Effects of ZIM3-KRAB domain on CRISPRi-induced tyr knockdown in embryos of X. tropicalis. (A) Schematic illustration of the experimental setup used to analyze the capacity of KRAB domain from Zim3 gene (ZIM3K) to target exogenous reporter gene in X. tropicalis embryos. The gTry13 were injected into the fertilized eggs together with dCas9-KM or dCas9-ZIM3K mRNAs, followed by embryo evaluation at 48 hpi as follows. (B) The qPCR validation of Tyr expression in the injected embryos. Data are presented as mean ± SEM (n = 5 per group). ****p < 0.0001 versus control group (one-way ANOVA test). Ns, no significant differences. (C) Representative images of tyrosinase expression in embryos co-injected with gTry13 and dCas9-mediated repressors at 48 hpi. The top panel shows low magnification (Scale bar = 1 mm), and the lower panel shows high magnification (Scale bar = 500 μm). (D) Embryos with different phenotypes were counted and compared with the total developed ones after injection. Total embryos evaluated for each group (n) is shown above each column.
	Fig. 1. Effects of PspCas13b and RfxCas13d on reporter gene expression in embryos of X. tropicalis. (A) Schematic of Cas13-related gRNAs targeting the mRNA of EGFP reporter gene. (B) Schematic illustration of the experimental setup used to analyze PspCas13b and RfxCas13d capacities to target exogenous reporter gene in X. tropicalis embryos. The mRNA of mCherry was used as the internal control. (C and D) Representative images (C) and quantification (C) of EGFP fluorescence in control and experimental embryos injected with PspCas13b system at 15 hpi (n = ~ 30 embryos from 3 independent experiments). (E and F) Representative images (E) and quantification (F) of EGFP fluorescence in control and experimental embryos injected with PspCas13b system at 24 hpi (n = ~ 30 embryos from 3 independent experiments). (G) The qPCR validation of EGFP expression in control and experimental embryos injected with PspCas13b system at 24 dpi (n = 5). (H and I) Representative images (H) and quantification (I) of EGFP fluorescence in control and experimental embryos injected with RfxCas13d system at 15 hpi (n = ~ 30 embryos from 3 independent experiments). (J and K) Representative images (J) and quantification (K) of EGFP fluorescence in control and experimental embryos injected with RfxCas13d system at 24 hpi (n = ~ 30 embryos from 3 independent experiments). (L) The qPCR validation of EGFP expression in control and experimental embryos injected with RfxCas13d system at 24 dpi (n = 5). All data are presented as mean ± SEM. Scale bar = 500 μm (C, E, and J) and 750 μm (H). Ns, no significant differences
	Fig. 2. Effects of PspCas13b and RfxCas13d on tyrosinase expression in embryos of X. tropicalis. (A) Schematic of Cas13-related gRNAs targeting the mRNA of endogenous tyrosinase (tyr) gene. (B) Schematic illustration of the experimental setup used to analyze the capacities of PspCas13b and RfxCas13d systems (300 pg/embryo) together with gTyr1-3 (100 pg/embryo). (C and D) Representative images (C) and quantification (D) of Tyr expression in control and experimental embryos injected with PspCas13b system at 24 hpi. Scale bar = 500 μm. (E and F) Representative images (E) and quantification (F) of Tyr expression in control and experimental embryos injected with PspCas13b system at 48 hpi. Scale bar = 1 mm. (G and H) Representative images (G) and quantification (H) of Tyr expression in control and experimental embryos injected with RfxCas13d system at 24 hpi. Scale bar = 500 μm. (I and J) Representative images (I) and quantification (J) of Tyr expression in control and experimental embryos injected with RfxCas13d system at 48 hpi. Scale bar = 1 mm. All data are presented as mean ± SEM (n = 4 per group). Ns, no significant differences
	Fig. 3. Effects of dCas9-KM system on reporter gene expression in embryos of X. tropicalis. (A) Schematic of CRISPRi-related gRNAs targeting the regions of mCherry reporter gene in pmCherry-N1 plasmid. (B) Schematic illustration of the experimental setup used to analyze dCas9-KM capacity targeting exogenous reporter gene in X. tropicalis embryos. (C) The qPCR validation of mCherry expression in embryos co-injected with dCas9-KM system and indicated gRNAs at 48 dpi (n = 4 per group). (D and E) Representative images (D) and quantification (E) of mCherry expression in embryos co-injected with dCas9-KM system and indicated gRNA at 48 hpi (n = ~ 60 embryos from 3 independent experiments). (F) Schematic of gR-187 targeting mCherry reporter gene in pCMV-mCherry-EF1α-EGFP dual-reporter plasmid. (G) The qPCR validation of mCherry expression in embryos co-injected with dCas9-KM system and indicated gRNA at 48 dpi (n = 5 per group). EGFP was used as the internal control. (H and I) Representative images (H) and quantification (I) of mCherry fluorescence in embryos co-injected with dCas9-KM system and indicate gRNA at 48 hpi (n = 24 embryos from 3 independent experiments). All data are presented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 versus control. Ns, no significant differences versus control
	Fig. 4. Effects of dCas9-KM system on tyrosinase expression in embryos of X. tropicalis. (A) Schematic illustration of dCas9-KM system-related gRNAs targeting the DNA locus of endogenous tyrosinase (Tyr) gene. Fertilized eggs were co-injected with dCas9-KM mRNAs and indicated gRNA sets, followed by embryo evaluation at 48 hpi as follows. (B and C) Representative images (B) and quantification (C) of Tyr production in embryos co-injected with dCas9-KM mRNA and indicated gRNAs at 48 hpi (n = 26 ~ 30 embryos from 3 independent experiments). Scale bar = 250 μm. (D) The qPCR validation of Tyr expression in embryos co-injected with dCas9-KM mRNA and indicated gRNAs at 48 dpi (n = 5 per group). (E and F) Representative images (E, scale bar = 250 μm) and quantification (F) of embryos with different phenotypes were counted and compared with the total developed ones 48 h post co-injection of dCas9-KM system. (G and H) Representative images (G) and quantification (H) of embryos with different phenotypes were counted and compared with the total developed ones 72 h post co-injection of dCas9-KM system. Total embryos evaluated for each group (n) is shown above each column. All data are present as mean ± SEM. ****p < 0.0001 versus gNC group
	Fig. 5. Effects of dCas9-KM system on pax6 expression in embryos of X. tropicalis. (A) Schematic illustration of dCas9-KM system-related gRNAs targeting the DNA locus of endogenous pax6 gene. Fertilized eggs were co-injected with dCas9-KM mRNAs and indicated gRNA sets, followed by embryo evaluation at 48 hpi as follows. (B) The qPCR validation of pax6 expression in the dCas9-KM-injected embryos with different gRNA sets (n = 5 per group). (C) Representative images of embryos with smaller eyes in the experimental group injected with pax6 targeting gRNA sets. The top panel shows low magnification, and the lower panel shows high magnification. Asterisk denotes smaller eyes. (D) Embryos with different phenotypes were counted and compared with the total developed ones after injection. Total embryos evaluated for each group (n) is shown above each column. (E) Quantification of eye size in tadpoles treated with gRNA sets and controls (n = 3 per group). (F) The qPCR validation of pax6 expression in the dCas9-KM-injected embryos with single gRNAs from set2 (n = 5 per group). All data are present as mean ± SEM. p < 0.05, p < 0.01, **p < 0.001 versus gNC group. Ns, no significant differences versus gNC group
	Fig. 6. Dose effect of dCas9-KM system on the efficiency of Tyr knockdown in X. tropicalis embryos. (A) Representative images (left and upper panels) and qPCR validation (right and lower panel) of tyrosinase expression in embryos co-injected with gTry13 and dCas9-KM mRNAs at 48 hpi. Fertilized eggs were co-injected with gTry13 (200 pg/embryo) and dCas9-KM mRNAs with different concentrations from 0 to 600 pg/embryo. Data are presented as mean ± SEM (n = 5 per group). p < 0.01, p < 0.001, **p < 0.0001 versus control group (one-way ANOVA test). Ns, no significant differences. Scale bar = 2500 μm. (B) Representative images (left and upper panels) and qPCR validation (right and lower panel) of tyrosinase expression in embryos co-injected with gTry13 and dCas9-KM mRNAs at 48 hpi. Fertilized eggs were co-injected with dCas9-KM mRNAs (300 pg/embryo) and gTry13 with different concentrations from 0 to 600 pg/embryo. Data are presented as mean ± SEM (n = 5 per group). p < 0.05, ****p < 0.0001 versus control group (one-way ANOVA test). ####p < 0.0001 (one-way ANOVA test). Ns, no significant differences. Scale bar = 1000 μm. (C and D) Representative images (C) and quantification (D) of embryos with different phenotypes were counted and compared with the total developed ones 48 hpi. Scale bar = 750 μm
	Fig. 7. Effects of ZIM3-KRAB domain on CRISPRi-induced tyr knockdown in embryos of X. tropicalis. (A) Schematic illustration of the experimental setup used to analyze the capacity of KRAB domain from Zim3 gene (ZIM3K) to target exogenous reporter gene in X. tropicalis embryos. The gTry13 were injected into the fertilized eggs together with dCas9-KM or dCas9-ZIM3K mRNAs, followed by embryo evaluation at 48 hpi as follows. (B) The qPCR validation of Tyr expression in the injected embryos. Data are presented as mean ± SEM (n = 5 per group). ****p < 0.0001 versus control group (one-way ANOVA test). Ns, no significant differences. (C) Representative images of tyrosinase expression in embryos co-injected with gTry13 and dCas9-mediated repressors at 48 hpi. The top panel shows low magnification (Scale bar = 1 mm), and the lower panel shows high magnification (Scale bar = 500 μm). (D) Embryos with different phenotypes were counted and compared with the total developed ones after injection. Total embryos evaluated for each group (n) is shown above each column