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Chimeric restriction enzymes: what is next?

机译：嵌合限制酶：下一步是什么？

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摘要

Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of Fokl (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes (Carroll et al., 1999). The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.
机译：嵌合限制性酶是一类新型的工程核酸酶，其中Fok1的非特异性DNA切割域（一种IIS限制性核酸内切酶）与其他DNA结合基序融合。后者包括三个常见的真核DNA结合基序，即螺旋-转-螺旋基序，锌指基序和含有亮氨酸拉链基序的基本螺旋-环-螺旋蛋白。这类嵌合核酸酶已显示出在体外进行非常接近预期识别序列的特异性切割。最重要的嵌合核酸酶是基于锌指DNA结合蛋白的酶，因为它们具有模块化的结构。最近，一种嵌合的核酸酶Zif-QQR-F（N）被证明可以在体内发现并裂解其靶标。这是通过将DNA底物和酶显微注射到青蛙卵母细胞中来测试的（Carroll等，1999）。即使将DNA组装成染色质后，注入的酶也会在靶标中产生位点特异性双链断裂。另外，这种切割激活了靶分子以进行有效的同源重组。由于锌指的识别特异性可以通过实验操作，因此可以设计嵌合核酸酶以靶向基因组内的特定位点。此类工程化的嵌合限制酶的可用性应使进行基因组工程（通常也称为基因疗法）成为可能。

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