德国德累斯顿工业大学Frank Buchholz团队，研发出通过锌指结构域插入激活特定DNA位点的重组酶。相关论文于2024年1月31日发表在《自然—生物技术》杂志上。

研究人员试图利用锌指DNA结合结构域（ZFDs）对重组酶的结合进行编程，通过产生融合蛋白将ZFDs插入到重组酶编码的序列中。通过筛选杂交蛋白文库，研究人员优化了插入位点、连接体长度、间距和ZFD方向，并生成了Cre型重组酶。在非融合的ZFD插入与重组酶结合位点附近的目标位点之前这种重组酶会保持休眠状态。

研究人员研发的融合技术将重组酶的定向编辑效率提高了四倍，并消除了哺乳动物细胞中可测量的脱靶活性。ZFD依赖的活性可转移到具有广谱特异性的重组酶中，为开发完全可编程的重组酶提供了手段。该研究提供了利用工程重组酶进行更精确、更高效基因组编辑的工具。

据了解，与核酸酶和其他编辑酶相比，重组酶作为基因组编辑工具具有一定的潜在优势，但将其工程化以高效重组特定DNA靶点的过程需要投入大量的时间和人力。

附：英文原文

Title: Activation of recombinases at specific DNA loci by zinc-finger domain insertions

Author: Mukhametzyanova, Liliya, Schmitt, Lukas Theo, Torres-Rivera, Julia, Rojo-Romanos, Teresa, Lansing, Felix, Paszkowski-Rogacz, Maciej, Hollak, Heike, Brux, Melanie, Augsburg, Martina, Schneider, Paul Martin, Buchholz, Frank

Issue&Volume: 2024-01-31

Abstract: Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site. The developed fusion improved targeted editing efficiencies of recombinases by four-fold and abolished measurable off-target activity in mammalian cells. The ZFD-dependent activity is transferable to a recombinase with relaxed specificity, providing the means for developing fully programmable recombinases. Our engineered recombinases provide improved genome editing tools with increased precision and efficiency.

DOI: 10.1038/s41587-023-02121-y

Source: https://www.nature.com/articles/s41587-023-02121-y