Production of Lentiviral Vectors Encoding the CRISPR-Cas13d System for RNA Targeting

The CRISPR-Cas13d system has been shown to be a potent tool for target RNA knockdown, offering advantages over DNA-editing systems by providing a non-genotoxic method to modulate gene expression. Efficient delivery of Cas13d and its guide RNAs into target cells is essential to achieve a strong knockdown effect, particularly in cell lines that are difficult to transfect and primary cells. Lentiviral vectors (LVs) are ideal for delivering large gene-editing tools due to their large packaging capacity and ability to transduce both dividing and nondividing cells. Here, we describe a detailed protocol for the production of high-titer, third-generation LVs with a dual-expression cassette for Cas13d and a customizable guide RNA. The method describes the cloning of target-specific guide RNAs, the transfection of HEK293T cells with packaging and transfer plasmids, the harvest and concentration of viral supernatants, and the determination of viral titer. The generated lentiviral particles can be used for stable transduction and efficient RNA knockdown in a broad range of mammalian cell types.