Journal: Molecular Therapy. Nucleic Acids

Article Title: Protection of animals against devastating RNA viruses using CRISPR-Cas13s

doi: 10.1016/j.omtn.2024.102235

Figure Lengend Snippet: Schematic workflow of the used approach in this study to inhibit FMDV infection using the CRISPR-Cas system (1) Transmission and circulation of FMD disease between wildlife-livestock ecosystems due to animal interactions. (2) Finding conserved regions among different FMDV serotypes by considering multiple criteria and designing efficient and specific crRNAs by the CaSilico tool. (3) Designed crRNAs of type VI-A/B/D were synthesized as DNA oligos, and then, each crRNA was cloned into separate crRNA cloning backbones. In addition to crRNA designing, several shRNAs were designed to compare virus knockdown by RNAi and CRISPR-Cas systems. (4) Virus propagation and titration of two FMDV serotypes (O and A) were performed on BHK-21 cells to be used for viral challenge. (5) The sgRNA screening was performed to validate potent crRNAs. (6) Two methods, RT-qPCR and TCID50 assay were used to measure viral RNA and FMDV infectivity, respectively. (7) CRISPR-Cas systems can potentially be leveraged to aid antiviral development for viral diseases in animals. Figure was created with BioRender.com .

Article Snippet: To generate crRNAs and shRNAs, spacers, shRNAs, and their reverse complementary DNA oligomers were synthesized as single-stranded DNA (ssDNA) by GenScript Biotech.

Techniques: Infection, CRISPR, Transmission Assay, Synthesized, Clone Assay, Cloning, Virus, Knockdown, Titration, Quantitative RT-PCR, TCID50 Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Protection of animals against devastating RNA viruses using CRISPR-Cas13s

doi: 10.1016/j.omtn.2024.102235

Figure Lengend Snippet: Effect of Cas13 and RNAi on FMDV inhibition, serotype O (A) Experimental procedure for evaluating Cas13s and RNAi antiviral activity in BHK-21 cells. BHK-21 cells were transfected with plasmids containing targeting and non-targeting shRNAs or Cas13 and crRNAs that target or do not target FMDV 24 h before infection. Then the supernatants were collected 24 hpi to evaluate the antiviral activity. Silencing efficiency of Lwa Cas13a (B), Psp Cas13b (C), Rfx Cas13d (D), and RNAi system (E) on FMDV virus according to RT-qPCR results. In (B) to (E), data points in the graph are five independent biological experiments performed in technical replicates, lines indicate means; error bars represent SD; t test was performed to compare each treatment to non-targeting crRNA or shRNA (green and red color of the boxes indicate p < 0.05 and p < 0.01, respectively).

Article Snippet: To generate crRNAs and shRNAs, spacers, shRNAs, and their reverse complementary DNA oligomers were synthesized as single-stranded DNA (ssDNA) by GenScript Biotech.

Techniques: Inhibition, Activity Assay, Transfection, Infection, Virus, Quantitative RT-PCR, shRNA

Journal: Oncotarget

Article Title: EglN2 contributes to triple negative breast tumorigenesis by functioning as a substrate for the FBW7 tumor suppressor

doi: 10.18632/oncotarget.14290

Figure Lengend Snippet: A, B, C . qRT-PCR (A), representative images (B) and quantification (C) of soft agar assays from C3Tag cells infected with the lentivirus encoding EglN2 shRNAs (744, 746) or Control (Ctrl) shRNA. The statistical significance was calculated using student's t test. *** denotes p value of < 0.005. Error bars represent one SEM. D, E . Representative tumor gross appearance and tumor weight plots for C3Tag cells infected with the lentivirus encoding EglN2 shRNA (744) or Control (Ctrl) shRNA implanted into the mammary fat pads in FVB/NJ mice. * denotes p value of <0.05 for comparison between two groups by using unpaired two sample t-test. Error bars represent one SEM. F . Kaplan-Meier survival curves for C3Tag: EglN2 +/+ and C3Tag: EglN2 −/− mice. The p value was determined by the Log-rank (Mantel-Cox) test.

Article Snippet: Lentiviral shRNAs were obtained from Broad Institute TRC shRNA library.

Techniques: Quantitative RT-PCR, Infection, Control, shRNA, Comparison