Tapping natural reservoirs of homing endonucleases for targeted gene modification.
|Title||Tapping natural reservoirs of homing endonucleases for targeted gene modification.|
|Publication Type||Journal Article|
|Year of Publication||2011|
|Authors||Takeuchi R, Lambert AR, Mak AN-S, Jacoby K, Dickson RJ, Gloor GB, Scharenberg AM, Edgell DR, Stoddard BL|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Date Published||2011 Aug 9|
|Keywords||Amino Acid Sequence, Base Sequence, Endonucleases, Gene Targeting, Humans, Models, Molecular, Molecular Sequence Data, Monoamine Oxidase, Mutagenesis, Protein Binding, Protein Engineering, Substrate Specificity|
Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for "genome editing" in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.
|Alternate Journal||Proc. Natl. Acad. Sci. U.S.A.|