This post was contributed by guest blogger, Kristian Laursen from Cornell University.
Site directed mutagenesis is a highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters/enhancers in reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome editing. Tailored mutations are commonly introduced to endogeneous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires a template of high homology to the endogenous target, yet to facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage.