Targeted Human Editing Using CRISPR Technology in Neural Stem Cells

Development of the programmable nuclease Ca9 has increased our understanding of the molecular basis of the physiological processes and diseases demonstrating a remarkable potential across various fields, including basic research, applied biotechnology, and biomedical research. CRISPR-Cas9 is a gene-editing technique that uses a guide RNA to direct the Cas9 enzyme to specific DNA sequences, where it creates double-strand breaks. These breaks can be repaired through either nonhomologous end joining (NHEJ), leading to gene mutations, or homology-directed repair (HDR), enabling precise edits. These characteristics render Cas9 an ideal candidate for various genome editing applications, particularly for the generation of knockout cells to study gene function either in vitro or in vivo. Delivery of the CRISPR molecular machinery is a crucial step for successful editing. Lentiviral vectors have been shown as an extremely effective method to transduce a wide range of cell types including neurons, astrocytes, adult neuronal stem cells, and oligodendrocytes. Here, we provide a useful protocol based on the lentiviral approach to develop CRISPR edition, facilitating the study of gene function in mammalian cells.