Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Adeno-associated viral vector resource for the RNA-targeting Cas13d: A comparison of high-fidelity variants, DjCas13d and hfCas13d

doi: 10.1016/j.omtm.2025.101565

Figure Lengend Snippet: AAVs encoding Cas13d can knock down targets in vitro and in vivo (A) The EFS-Cas13d-mCherry ( A) and the CMV-intron-Cas13d ( A) encoding AAVs were used to transduce 293FT cells in culture. Seventy-two hours later, the cells were harvested and Cas13d protein levels were examined via western blotting with an anti-HA tag antibody. (B) Only the cells that received the CMV-intron encoding Cas13d AAVs exhibited detectable expression of the HA-tagged Cas13d protein. B-tubulin levels were used as a loading control. n = 1. (C) CMV-intron encoding Cas13d AAVs, an AAV encoding a GFP gene, and AAVs either encoding a gRNA expression cassette designed to knockdown GFP or LacZ as a control were used to transduce 293FT cells in culture. Seventy-two hours later, the cells were harvested and qPCR was performed to assess GFP knockdown. (D) All three Cas13d encoding AAVs, (CasRx, hfCas13d, and DjCas13d) were capable of significantly reducing GFP mRNA levels, n = 4. (E) An AAV encoding CMV-DjCas13d and an AAV encoding a GFP gene with either a gRNA expression cassette designed to target BDNF or LacZ as a control were co-injected into the rat IL cortex and 3 weeks later, qPCR and IHC were performed. (F) Quantitative PCR revealed BDNF was significantly knocked down within the IL cortex compared to the LacZ control group, (∗ p < 0.05). LacZ gRNA group, n = 6; BDNF gRNA group, n = 5). Error bars = standard error of the mean. (G) The image on the left depicts a hemisected coronal brain slice taken from an animal that received the injection of viruses described (E and F). IL, infralimbic cortex; PrL, prelimbic cortex; M1, primary motor cortex. The location of viral transduction in the IL is obvious by visualizing native GFP fluorescence. Images to the right (i–vi) depict two magnified views from the brain slice and stained for DAPI, GFP fluorescence, and anti-HA DjCas13d expression IHC. Boxes to the top (i–iii) are from a control region from the M1 motor cortex that was not injected with virus. Boxes on the bottom (iv–vi) are magnified views of the viral transduction site. Scale bars are shown in vi., 200 microns. (D) Two-way ANOVA followed by Tukey post-hoc, ∗ p < 0.05. (F) Independent t test, ∗ p < 0.05. Error bars = standard error of the mean.

Article Snippet: Then, the blocking solution was replaced by a 120 μL blocking solution containing the primary antibody at a 1:800 dilution (Cell Signaling HA-tag C29F4 rabbit mAB3724).

Techniques: Knockdown, In Vitro, In Vivo, Transduction, Western Blot, Expressing, Control, Injection, Real-time Polymerase Chain Reaction, Slice Preparation, Fluorescence, Staining, Virus