Directed evolution allows the effective engineering of proteins, biosynthetic pathways, and cellular functions. Traditional plasmid-based methods generally subject one or occasionally multiple genes-of-interest to mutagenesis, require time-consuming manual interventions, and the genes that are subjected to mutagenesis are outside of their native genomic context. Other methods mutagenize the whole genome unselectively which may distort the outcome. Recent recombineering- and CRISPR-based technologies radically change this field by allowing exceedingly high mutation rates at multiple, predefined loci in their native genomic context. In this review, we focus on recent technologies that potentially allow accelerated tunable mutagenesis at multiple genomic loci in the native genomic context of these target sequences. These technologies will be compared by four main criteria, including the scale of mutagenesis, portability to multiple microbial species, off-target mutagenesis, and cost-effectiveness. Finally, we discuss how these technical advances open new avenues in basic research and biotechnology.

中文翻译：

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微生物中多个染色体区域的靶向诱变。

定向进化允许对蛋白质、生物合成途径和细胞功能进行有效的工程改造。传统的基于质粒的方法通常对一个或偶尔多个感兴趣的基因进行诱变，需要耗时的手动干预，并且进行诱变的基因超出其天然基因组环境。其他方法非选择性地诱变整个基因组，这可能会扭曲结果。最近基于重组工程和 CRISPR 的技术通过允许在其天然基因组环境中的多个预定义位点具有极高的突变率，从根本上改变了这一领域。在这篇综述中，我们关注最近的技术，这些技术可能允许在这些靶序列的天然基因组环境中的多个基因组位点加速可调诱变。这些技术将通过四个主要标准进行比较，包括诱变的规模、对多种微生物物种的可移植性、脱靶诱变和成本效益。最后，我们讨论了这些技术进步如何为基础研究和生物技术开辟新途径。