Abstract

CRISPR-Cas9 is a technique used for genome editing that is adapted from bacterial antiviral immune mechanisms. This CRISPR-Cas9 system has been optimized for use in mammalian cells. By introducing a single-guide RNA sequence (sgRNA) specific for our gene of interest, we can specifically recruit Cas9 to this gene. The nuclease introduces a double stranded break into the DNA that is repaired usually repaired through Non-Homologous End Joining (NHEJ) in mammalian cells. This repair pathway can knock-out the gene of interest by introducing frame shift mutations. Alternatively, knock-in mutations can be generated by additionally providing a template for Homologous Recombination (HR). A knock-in can either be a single nucleotide substitution or an extended sequence encoding a full protein. This is an effective approach to study known protein mutations, to screen for mutations that affect protein function, or add tags to endogenous proteins in cell lines, in addition to other applications. Knock-out cells are used to study the biological function of a gene, to generate disease models, to generate unique phenotypes, such as MHC-deficient cells, to identify and validate therapeutic targets, to identify factors for viral entry, and much more.