Journal: Molecular Therapy. Nucleic Acids

Article Title: CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9

doi: 10.1016/j.omtn.2025.102556

Figure Lengend Snippet: Dual-gRNA SaCas9 construct targeting JCV LT-Ag and VP1 produces the greatest reduction in JCV viral load (A) Map depicting optimal SaCas9-compatible gRNA targets on circular dsDNA genome of Mad-1 per bioinformatic software Benchling . Targeted regions include the NCCR (black), early transcript LT-Ag (red), and late transcript VP1 (blue). Seven separate combinations of these gRNAs were incorporated into SaCas9-construct, pPapi, to determine the most effective antiviral. (B) Western blot depicting viral protein levels of cells treated with CRISPR constructs described above. Compared to untreated control, the largest reduction in both VP1 (42 kD) and Agno (11 kD) is evident for cells treated with the two-gRNA construct targeting LT-Ag and VP1 (LTAg+VP1). (C) Viral genomic copy number measured by qPCR of DNA extracted from cell lysates of conditions described above reveals a statistically significant decrease in cells expressing the LTAg+VP1 dual-gRNA construct compared to untreated control (∗ = p = 0.0229, n = 16). (D) Viral genomic copy number measured by qPCR of DNA extracted from media of cell conditions described above reveals a statistically significant decrease in the media of the LTAg+VP1 dual-gRNA cells compared to untreated control (∗ p = 0.0318, n = 16).

Article Snippet: Using Benchling bioinformatic software, we designed three gRNAs and compared the efficacy of seven potential combinations with SaCas9.

Techniques: Construct, Software, Western Blot, CRISPR, Control, Expressing