Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North A3-025, Seattle, WA 98109, USA.
Proc Natl Acad Sci U S A. 2011 Aug 9;108(32):13077-82. doi: 10.1073/pnas.1107719108. Epub 2011 Jul 22.
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.
归巢内切核酸酶通过在潜在宿主 DNA 中的单个靶位产生双链断裂来动员其自身基因。由于其高度特异性，这些蛋白质被用于高等真核生物的“基因组编辑”。然而，改变归巢内切核酸酶的特异性是相当具有挑战性的。在这里，我们描述了 200 多种天然 LAGLIDADG 归巢内切核酸酶（LHE）的鉴定和系统发育分析。对系统发育树中一个分支内的内切核酸酶的生化和结构特征进行了研究，结果表明蛋白质结构高度保守，而 DNA 靶位高度分化，并表明这些蛋白质中有很大一部分具有足够的稳定性和活性，可以作为工程支架。利用这一信息，我们创造了一种靶向酶，用于破坏人细胞内的内源性单胺氧化酶 B 基因。本研究中描述的 LHE 的普遍存在和多样性可能有助于为基因组编辑创建许多定制的核酸酶。
