Characterizing the portability of phage-encoded homologous recombination proteins.
Characterizing the portability of phage-encoded homologous recombination proteins.
复制标题
DOI：
10.1038/s41589-020-00710-5
复制
发表时间：
2021-04
影响因子：
14.8
通讯作者：
Church GM
中科院分区：
文献类型：
--
作者：
Filsinger GT;Wannier TM;Pedersen FB;Lutz ID;Zhang J;Stork DA;Debnath A;Gozzi K;Kuchwara H;Volf V;Wang S;Rios X;Gregg CJ;Lajoie MJ;Shipman SL;Aach J;Laub MT;Church GM
高效的基因组编辑方法对于生物技术和基础研究至关重要。同源重组（HR）是基因组编辑的最通用方法，但依赖于宿主RecA介导的途径的技术效率低下且费力。噬菌体编码的ssDNA退火蛋白（SSAPs）提高HR 1000倍以上的内源性水平;然而，他们不是广泛的功能。使用大肠杆菌，乳酸乳球菌，耻垢分枝杆菌，鼠李糖乳杆菌，新月柄杆菌，我们调查了有限的可移植性的SSAPs。我们发现，这些蛋白质特异性地识别宿主的单链DNA结合蛋白（SSB）的C-末端尾部，并且如果与该宿主结构域的兼容性得以维持，则在物种之间是便携式的。此外，我们发现，与配对的SSB共表达SSAP可以显着提高活性，在一些物种中，即使没有宿主相容性，也能实现SSAP功能。最后，我们发现，高效率的人力资源远远超过了常用的随机诱变方法的突变能力，产生特殊的表型无法通过连续的核苷酸转换。
Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded ssDNA annealing proteins (SSAPs) improve HR 1000-fold above endogenous levels; however, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host’s single-stranded DNA-binding protein (SSB), and are portable between species if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with a paired SSB can significantly improve activity, in some species enabling SSAP functionality even without host-compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes inaccessible through sequential nucleotide conversions.