Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Pseudoknot-targeting Cas13b combats SARS-CoV-2 infection by suppressing viral replication.

doi: 10.1016/j.ymthe.2023.03.018

Figure Lengend Snippet: Figure 1. mRNA-encoded Cas13b strongly blocks SARS-CoV-2 genomic replication by targeting the RdRp-coding region (A) Sequence diversity of the coronaviruses SARS-CoV-2, SARS-CoV, and MERS-CoV. SARS-CoV-2 complete genomes derived from patients were aligned to calculate the percentage identity. Twelve candidate RdRp-targeting crRNAs are indicated in the lower panel. (B) Luciferase activity assay showing the RdRp-targeting efficiency of the 12 crRNAs. HEK293T cells, expressing PspCas13b and crRNA, were transfected with luciferase vectors conjugated with RdRp fragments. For the dual-luciferase assay, the cells were harvested after 24 h (n = 3 for each group). The firefly luminescence is normalized by Renilla luminescence. One-way analysis of variance (ANOVA) was used for statistical analysis. ns, not significant; ***p < 0.0001. (C) Representative images of the spike protein (green)- and DAPI (blue)-stained Vero E6 cells. SARS-CoV-2 Hu-1 obtained from cells and the supernatant were used to re-infected Vero E6 cells transfected with Cas13b mRNA and crRNAs 24 h before infection. After 24 h, the in- fected cells were stained and visualized under a fluorescent microscope. Scale bars, 100 mm. (D) Quantification for the percentage of DAPI- and spike protein-positive cells, tested as in (C). (E) Quantification of SARS-CoV-2 gene copies. The viral RNA level, tested as in (C), was quantified by qRT-PCR (n = 3 for each group). (F) Titration of the virus obtained in (C). Vero E6 cells were infected by the viruses. After 72 h of infection, the number of plaques was measured and multiplied by the dilution factor to calculate the virus titer (n = 3 for each group). Values are means ± SEM. Red scores in (E and F) indicate the reduction percentage of gene copies and virus titer, respectively. SARS-CoV, severe acute respiratory syndrome coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; RdRp, RNA-dependent RNA polymerase; crRNA, CRISPR RNA; DAPI, 40,6-diamidino-2-phenylindole; qRT-PCR, quantitative reverse transcription PCR; SEM, standard error of the mean.

Article Snippet: The crRNA plasmids were modified from PspCas13b crRNA backbone vector (Addgene, plasmid 103854) by BbsI restriction and T4 ligation cloning.

Techniques: Sequencing, Derivative Assay, Luciferase, Activity Assay, Expressing, Transfection, Staining, Infection, Microscopy, Quantitative RT-PCR, Titration, Virus, CRISPR, Reverse Transcription

Journal: Nature Communications

Article Title: N 6 -methyladenosine regulates glycolysis of cancer cells through PDK4

doi: 10.1038/s41467-020-16306-5

Figure Lengend Snippet: a Schematic representation of positions of m 6 A site within PDK4 mRNA and the regions targeted by three gRNAs, respectively. b The threshold cycle (Ct) of qPCR showing SELECT results for detecting m 6 A site in the potential m 6 A site of PDK4 5′UTR in Mettl3 Mut/- HeLa, sh-Mettl3 Huh7 and their corresponding control cells. c The threshold cycle (Ct) of qPCR showing SELECT results for detecting m 6 A site in 5′UTR of PDK4 in HeLa cells transfected with dCas13b-ALKBH5 combined with gRNA control or gRNA1/2/3, respectively, for 24 h. d , e The mRNA ( d ) or protein ( e ) expression of PDK4 in HeLa cells transfected with dCas13b-ALKBH5 combined with gRNA control or gRNA1/2/3, respectively, for 24 h. f RIP-qPCR analysis of PDK4 mRNA in HeLa cells transfected with dCas13b-ALKBH5 combined with gRNA control (dC-A5) or gRNA for PDK4 (dC-A5 + gRNA) for 24 h by use of antibodies against YTHDF1 and IGF2BP3, respectively. g HeLa cells were transfected with gRNA control, gRNA1 for PDK4, and dCas13b-ALKBH5 for 24 h and then further treated with Act-D for the indicated times. The mRNA of PDK4 was checked by qRT-PCR. h – j The glucose consumption ( h ), lactate production ( i ), and ATP levels ( j ) in HeLa cells transfected with gRNA control, gRNA1 for PDK4 , and dCas13b-ALKBH5 or dCas13b-ALKBH5-Mut for 24 h. Data are presented as means ± SD from three independent experiments. ** p < 0.01, NS, no significant, by two-tailed unpaired Student’s t test for ( b ) ( p = 0.001 and p = 0.002, respectively), ( h – j ) one-way ANOVA for ( c ) ( p < 0.001), ( d ) ( p < 0.001), and ( e ) ( p < 0.001), and two-way ANOVA for ( f ) and ( j ) ( p < 0.001).

Article Snippet: To generate dCas13b-ALKBH5 fusion protein, the original PspCas13b plasmid (Addgene plasmid #103866), gRNA plasmid (Addgene plasmid #103854) and non-targeting gRNA plasmid (Addgene plasmid #103868) were obtained from Addgene.

Techniques: Control, Transfection, Expressing, Quantitative RT-PCR, Two Tailed Test