guide rna ngrna backbones  (Addgene inc)

Journal: Cell & bioscience

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

doi: 10.1186/s13578-025-01397-8

Figure Lengend Snippet: 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 exog enous 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

Article Snippet: The human codon-optimized PspCas13b (pC0046, #103862) and RfxCas13d (pXR001, #109049) expressing plasmids as well as their guide RNA (gRNA) expression plasmids (pC0043, #103854 for PspCas13b; pXR003, #109053 for RfxCas13d) were purchased from Addgene.

Techniques: Gene Expression, Plasmid Preparation, Biomarker Discovery, Expressing, Injection, Control, Fluorescence

Journal: Cell & bioscience

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

doi: 10.1186/s13578-025-01397-8

Figure Lengend Snippet: 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

Article Snippet: The human codon-optimized PspCas13b (pC0046, #103862) and RfxCas13d (pXR001, #109049) expressing plasmids as well as their guide RNA (gRNA) expression plasmids (pC0043, #103854 for PspCas13b; pXR003, #109053 for RfxCas13d) were purchased from Addgene.

Techniques: Expressing, Injection, Biomarker Discovery