聚合酶引导的碱基编辑可实现体内诱变和快速蛋白质工程。
Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering.
机构信息
Department of Bioengineering, 443 Via Ortega, MC 4245, Stanford University, Stanford, CA, USA.
Department of Chemical Engineering, 443 Via Ortega, MC 4245, Stanford University, Stanford, CA, USA.
出版信息
Nat Commun. 2021 Mar 11;12(1):1579. doi: 10.1038/s41467-021-21876-z.
Random mutagenesis is a technique used to generate diversity and engineer biological systems. In vivo random mutagenesis generates diversity directly in a host organism, enabling applications such as lineage tracing, continuous evolution, and protein engineering. Here we describe TRIDENT (TaRgeted In vivo Diversification ENabled by T7 RNAP), a platform for targeted, continual, and inducible diversification at genes of interest at mutation rates one-million fold higher than natural genomic error rates. TRIDENT targets mutagenic enzymes to precise genetic loci by fusion to T7 RNA polymerase, resulting in mutation windows following a mutation targeting T7 promoter. Mutational diversity is tuned by DNA repair factors localized to sites of deaminase-driven mutation, enabling sustained mutation of all four DNA nucleotides at rates greater than 10 mutations per bp. We show TRIDENT can be applied to routine in vivo mutagenesis applications by evolving a red-shifted fluorescent protein and drug-resistant mutants of an essential enzyme.
随机诱变是一种用于产生多样性和工程生物系统的技术。体内随机诱变直接在宿主生物体内产生多样性，使其能够应用于谱系追踪、连续进化和蛋白质工程等领域。本文描述了 TRIDENT（通过 T7 RNA 聚合酶实现靶向体内多样化的技术），这是一种在目标基因上进行靶向、持续和诱导多样化的平台，其突变率比自然基因组错误率高一百万倍。TRIDENT 通过与 T7 RNA 聚合酶融合将诱变酶靶向到精确的遗传基因座，导致突变窗口紧随 T7 启动子的靶向突变之后。通过将 DNA 修复因子定位到脱氨酶驱动突变的位点，调节突变的多样性，从而能够以大于每碱基 10 个突变的速度持续突变所有四个 DNA 核苷酸。我们通过进化出一个红移荧光蛋白和一种必需酶的耐药突变体，展示了 TRIDENT 可以应用于常规的体内诱变应用。
Zotero 插件