Journal: Nucleic Acids Research

Article Title: Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

doi: 10.1093/nar/gkaa269

Figure Lengend Snippet: Design of dm 6 ACRISPR for targeted RNA demethylation. ( A ) Overview of site-specific RNA targeting using dCas13b-guided fusion proteins with gRNA close to or away from the target site; ( B ) Schematic representation of the domain organization of dCas13b-ALKBH5 or ALKBH5-dCas13b expression cassette; ( C ) Expression of dCas13b-ALKBH5 and ALKBH5-dCas13b fusion protein in HeLa cells measured by western blotting using ALKBH5 antibody.

Article Snippet: The original PspCas13b plasmid (Addgene plasmid #103866), gRNA plasmid (Addgene plasmid #103854) and non-targeting gRNA plasmid (Addgene plasmid #103868) were purchased from Addgene.

Techniques: Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

doi: 10.1093/nar/gkaa269

Figure Lengend Snippet: dm 6 ACRISPR induces demethylation of single m 6 A-modified mRNA. ( A ) Schematic representation of positions of the m 6 A site within CYB5A mRNA and regions targeted by three gRNAs; ( B ) m 6 A enrichment of CYB5A mRNA in HeLa and HEK293T cells was analyzed by m 6 A RIP-qPCR analysis using m 6 A antibody; ( C ) Threshold cycle (Ct) of qPCR showing SELECT results for detecting the m 6 A site in CYB5A at A48 in HeLa (wide-type) and HEK293T cells (sh-Control) compared to their corresponding METTL3-knockdown cells (M3KD) with fold changes listed; ( D ) mRNA expression of CYB5A in HEK293T cells cotransfected with wild-type (WT) Cas13b and NT-gRNA (control) or gRNA1/2/3 for 24 h; ( E, F ) Threshold cycle (Ct) of SELECT-qPCR detecting the m 6 A site in CYB5A at A48 in HEK293T cells transfected with dCas13b-ALKBH5 (E) or dCas13b-ALKBH5 H204A (F) combined with NT-gRNA or gRNA1/2/3, respectively, for 24 h, with fold changes listed; ( G ) mRNA levels of CYB5A in HEK293T cells transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2/3, respectively, for 24 h; ( H ) HEK293T cells were pre-transfected as indicated for 24 h and then further treated with Act-D for the indicated times. The mRNA levels of CYB5A were measured by qRT-PCR. oe, overexpression; ( I ) HEK293T cells were pre-transfected as indicated for 24 h. Binding between YTHDF2 and CYB5A mRNAs was checked by RIP-qPCR using YTHDF2 antibody. Data are presented as mean ± SD from three independent experiments. ** P < 0.01 by Student's t test (B and C) or one-way ANOVA (D, E, F, G, H and I).

Article Snippet: The original PspCas13b plasmid (Addgene plasmid #103866), gRNA plasmid (Addgene plasmid #103854) and non-targeting gRNA plasmid (Addgene plasmid #103868) were purchased from Addgene.

Techniques: Modification, Control, Knockdown, Expressing, Transfection, Quantitative RT-PCR, Over Expression, Binding Assay

Journal: Nucleic Acids Research

Article Title: Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

doi: 10.1093/nar/gkaa269

Figure Lengend Snippet: dm 6 ACRISPR induces demethylation of multiple m 6 A sites of 5′UTR. ( A ) Schematic representation of positions of m 6 A sites within CTNNB1 mRNA and regions targeted by three gRNAs; ( B ) Threshold cycle (Ct) of qPCR showing SELECT results for detecting m 6 A in three potential m 6 A sites of CTNNB1 in HEK293T cells transfected with vector control (pcDNA3.1) or pcDNA/ALKBH5, with fold changes listed; ( C , D ) Threshold cycle (Ct) of qPCR showing SELECT results for detecting the m 6 A S1 (C) or S2 (D) site in CTNNB1 in HEK293T cells transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2/3, respectively, for 24 h, with fold changes listed; ( E ) mRNA levels of CTNNB1 in HEK293T cells transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2/3, respectively, for 24 h; ( F ) HEK293T cells were pre-transfected as indicated for 24 h and then further treated with Act-D for the indicated time periods. The mRNA levels of CTNNB1 were measured by qRT-PCR. oe, overexpression; ( G ) HEK293T cells were pre-transfected as indicated for 24 h. Binding between YTHDF2 and CTNNB1 mRNA was checked by RIP-qPCR using YTHDF2 antibody; ( H ) Protein expression of β-catenin in HEK293T cells transfected with dCas13b-ALKBH5 combined with NT-gRNA (NT) or gRNA1/2/3, respectively, for 24 h was checked by western blot analysis ( left ) and quantitatively analyzed ( right ); ( I ) cells were transfected with pGL-5′UTR, NT-gRNA (NT) or gRNA1, and dCas13b-ALKBH5 or ALKBH5-dCas13b and pRL-TK reporter for 24 h. Translation efficiency is defined as the quotient of reporter protein production (F-luc/R-luc) divided by mRNA abundance . Data are presented as mean ± SD from three independent experiments. ** P < 0.01 by Student's t test (B and I) or one-way ANOVA (C, D, E, F, G and H).

Article Snippet: The original PspCas13b plasmid (Addgene plasmid #103866), gRNA plasmid (Addgene plasmid #103854) and non-targeting gRNA plasmid (Addgene plasmid #103868) were purchased from Addgene.

Techniques: Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Over Expression, Binding Assay, Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

doi: 10.1093/nar/gkaa269

Figure Lengend Snippet: Specificity and non-additive effects of dm 6 ACRISPR on m 6 A demethylation. ( A–C ) Cells were transfected with dCas13b-ALKBH5 and NT-gRNA or gRNA1/2/3 of CYB5A, respectively, for 24 h, with m 6 A levels of PRSS56 (A), GALR1 (B), or INPP4A (C) measured by m 6 A -RIP-qPCR analysis; ( D ) Smooth scatter plot displaying mean methylation in NT-gRNA (control) or CYB5A gRNA-1-transfected HEK293T cells for 24 h with dCas13b-ALKBH5 (total 25 888 peaks). Red dots indicate peaks with significant upregulation while blue dots indicate peaks with significant downregulation in methylation ( P < 0.05); ( E ) Smooth scatter plot displaying mean expression of transcripts in NT-gRNA (control) or CYB5A gRNA-1-transfected HEK293T cells for 24 h with dCas13b-ALKBH5 (total 25,603 transcripts). Red dots indicate transcripts with significant upregulation while blue dots indicate transcripts with significant downregulation ( P < 0.05); ( F , G ) HEK293T cells were transfected with si-NC or si-CYB5A-1 together with NT-gRNA (NT) or CYB5A gRNA-1 combined with dCas13b-ALKBH5 for 24 h. The m 6 A fold enrichment of ZNF581 (F) or EXOSC2 (G) was analyzed by m 6 A RIP-qPCR analysis, with fold changes listed; ( H , I ) cells were transfected with si-NC or si-CYB5A-1 together with NT-gRNA (NT) or CYB5A gRNA-1 for 24 h. Relative mRNA levels of PPAN (H) or MAPRE2 (I) were analyzed by qPCR analysis, with fold changes listed. Data are presented as mean ± SD from three independent experiments. ** P < 0.01, NS, no significant, by one-way ANOVA with Bonferroni test.

Article Snippet: The original PspCas13b plasmid (Addgene plasmid #103866), gRNA plasmid (Addgene plasmid #103854) and non-targeting gRNA plasmid (Addgene plasmid #103868) were purchased from Addgene.

Techniques: Transfection, Methylation, Control, Expressing

Journal: Nucleic Acids Research

Article Title: Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

doi: 10.1093/nar/gkaa269

Figure Lengend Snippet: Targeting m 6 A of oncogene transcripts by dm 6 ACRISPR regulates cell proliferation. ( A ) m 6 A RIP-qPCR analysis of EGFR mRNA in HeLa cells transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2, respectively, for 24 h; ( B ) Protein expression of EGFR in HeLa cells untransfected or transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2, respectively, for 24 h, and checked by western blot analysis ( left ) and quantitatively analyzed ( right ); ( C ) Proliferation of HeLa cells untransfected or transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2, respectively, for the indicated time periods; ( D ) m 6 A RIP-qPCR analysis of MYC mRNA in HeLa cells transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2, respectively, for 24 h; ( E ) Protein expression of MYC in HeLa cells untransfected or transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2, respectively, for 24 h, and checked by western blot analysis ( left ) and quantitatively analyzed ( right ); ( F ) Proliferation of HeLa cells untransfected or transfected with dCas13b-ALKBH5 combined with NT-gRNA or gRNA1/2, respectively, for the indicated time periods. Data are presented as mean ± SD from three independent experiments. ** P < 0.01, NS, no significant, by one-way ANOVA with Bonferroni test.

Article Snippet: The original PspCas13b plasmid (Addgene plasmid #103866), gRNA plasmid (Addgene plasmid #103854) and non-targeting gRNA plasmid (Addgene plasmid #103868) were purchased from Addgene.

Techniques: Transfection, Expressing, Western Blot