Site-directed mutagenesis

Since 2013, the development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has also allowed for the editing of the genome, and mutagenesis may be performed in vivo with relative ease.

[6] Site-directed mutagenesis was achieved in 1974 in the laboratory of Charles Weissmann using a nucleotide analogue N4-hydroxycytidine, which induces transition of GC to AT.

In 1971, Clyde Hutchison and Marshall Edgell showed that it is possible to produce mutants with small fragments of phage ΦX174 and restriction nucleases.

[9][10] Hutchison later produced with his collaborator Michael Smith in 1978 a more flexible approach to site-directed mutagenesis by using oligonucleotides in a primer extension method with DNA polymerase.

[11] For his part in the development of this process, Michael Smith later shared the Nobel Prize in Chemistry in October 1993 with Kary B. Mullis, who invented polymerase chain reaction.

A large number of methods are available to effect site-directed mutagenesis,[12] although most of them have rarely been used in laboratories since the early 2000s, as newer techniques allow for simpler and easier ways of introducing site-specific mutation into genes.

[13] The DNA fragment to be mutated is inserted into a phagemid such as M13mp18/19 and is then transformed into an E. coli strain deficient in two enzymes, dUTPase (dut) and uracil deglycosidase (udg).

Both enzymes are part of a DNA repair pathway that protects the bacterial chromosome from mutations by the spontaneous deamination of dCTP to dUTP.

These methods require multiple steps of PCR so that the final fragment to be ligated can contain the desired mutation.

Since 2013, the development of CRISPR-Cas9 technology has allowed for the efficient introduction of various mutations into the genome of a wide variety of organisms.

[21][22] Site-directed mutagenesis is used to generate mutations that may produce a rationally designed protein that has improved or special properties (i.e.protein engineering).

For instance changing a particular serine (phosphoacceptor) to an alanine (phospho-non-acceptor) in a substrate protein blocks the attachment of a phosphate group, thereby allows the phosphorylation to be investigated.

For example, commonly used laundry detergents may contain subtilisin, whose wild-type form has a methionine that can be oxidized by bleach, significantly reducing the activity the protein in the process.

[25] This methionine may be replaced by alanine or other residues, making it resistant to oxidation thereby keeping the protein active in the presence of bleach.

This method allows for extensive mutagenesis over multiples sites, including the complete redesign of the codon usage of gene to optimise it for a particular organism.

Depiction of one common way to clone a site-directed mutagenesis library (i.e., using degenerate oligos). The gene of interest is PCRed with oligos that contain a region that is perfectly complementary to the template (blue), and one that differs from the template by one or more nucleotides (red). Many such primers containing degeneracy in the non-complementary region are pooled into the same PCR, resulting in many different PCR products with different mutations in that region (individual mutants shown with different colors below).
Site saturation mutagenesis is a type of site-directed mutagenesis. This image shows the saturation mutagenesis of a single position in a theoretical 10-residue protein. The wild type version of the protein is shown at the top, with M representing the first amino acid methionine, and * representing the termination of translation. All 19 mutants of the isoleucine at position 5 are shown below.