DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors". The nucleases make specific double-strand breaks (DSBs) at desired places in the genome. The cell’s own mechanisms repair the induced break(s) by natural processes.
At present there are four families of engineered nucleases being used.[3][4][5][6]
To understand the function of a gene or a protein one interferes with it in a sequence-specific way, and watches its effects on the organism. However, in some organisms it is difficult or impossible to do site-specific mutation. Therefore more indirect methods have to be used. Examples are:
Genome editing with nucleases such as ZFN. This is different from siRNA. The engineered nuclease (the enzyme which cuts the DNA) is able to modify DNA-binding. This way it can (in principle) cut any targeted position in the genome, and introduce change in sequences for genes which cannot be specifically targeted by conventional RNAi.
Genome editing was chosen by Nature Methods as the 2011 Method of the Year.[8] The technique is already being used, but implanting modified embryos into a woman is not yet permitted.[9][10]
In 2017 this system was announced as one of the biggest scientific achievements of the year. Cas9 is an enzyme which, with a guide RNA, can put a new sequence of DNA into a genome. Sir John Skehel[11] said "That might allow you to knock out a particular gene in a cell, or introduce a particular gene, or correct a particular mutated gene that you want to work better".[12]
↑Puchta, H.; Fauser, F. (2013). "Gene targeting in plants: 25 years later". Int. J. Dev. Biol. 57 (6–7–8): 629–637. doi:10.1387/ijdb.130194hp. PMID24166445.
↑Fire, Andrew; Xu, Siqun; Montgomery, Mary K.; Kostas, Steven A.; Driver, Samuel E.; Mello, Craig C. (1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature. 391 (6669): 806–811. Bibcode:1998Natur.391..806F. doi:10.1038/35888. PMID9486653.