A repackaged CRISPR platform increases homology-directed repair for yeast engineering
A repackaged CRISPR platform increases homology-directed repair for yeast engineering
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DOI：
10.1038/s41589-021-00893-5
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发表时间：
2021-10-28
影响因子：
14.8
通讯作者：
Shao, Zengyi
中科院分区：
文献类型：
--
作者：
Ploessl, Deon;Zhao, Yuxin;Shao, Zengyi
低效的同源定向修复 (HDR) 限制了优先采用非同源末端连接 (NHEJ) 修复 DNA 双链断裂 (DSB) 的生物体中的 CRISPR-Cas9 基因组编辑。目前用于降低 NHEJ 熟练程度的策略涉及 NHEJ 破坏。为了在不破坏 NHEJ 的情况下实现精确编辑，我们发现了传统 CRISPR 平台的缺点，并开发了一种 CRISPR 平台降低的插入缺失核酸酶系统，可实现精确修复 (LINEAR)，与之前报告中在四种 NHEJ 熟练酵母中使用传统平台的报告相比，该系统提高了 HDR 率（至 67-100%）。通过保留 NHEJ，我们展示了其调查基因组景观的能力，识别其时空基因组结构为异源途径产生有利表达动态的位点。我们提出了一个案例研究，该研究部署了 LINEAR 精确编辑和 NHEJ 介导的随机整合，以快速设计和优化微生物工厂来生产 (S)-去甲乌酸。总而言之，这项工作展示了如何利用一对拮抗的 DNA DSB 修复途径来扩大当前微生物工厂的集合。
Inefficient homology-directed repair (HDR) constrains CRISPR-Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR)-which enhanced HDR rates (to 67-100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.