Dissecting quantitative trait nucleotides by saturation genome editing.
Dissecting quantitative trait nucleotides by saturation genome editing.
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通过饱和基因组编辑来剖析数量性状核苷酸。
DOI：
10.1101/2024.02.02.577784
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Steinmetz,LarsM
中科院分区：
文献类型：
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作者：
Roy,KevinR;Smith,JustinD;Li,Shengdi;Vonesch,SibylleC;Nguyen,Michelle;Burnett,WallaceT;Orsley,KevinM;Lee,Cheng-Sheng;Haber,JamesE;StOnge,RobertP;Steinmetz,LarsM
基因组编辑技术有可能通过遗传变异的系统工程和表型表征，改变我们对遗传变异如何产生复杂性状的理解。然而，目前还没有一个系统具有足够的效率、保真度和吞吐量来全面识别基因组规模的因果变异。在这里，我们探索了模板化CRISPR编辑系统在出芽酵母中安装全基因组自然变异的能力。我们优化了几种方法来增强与供体DNA模板的同源性定向修复（HDR），包括供体招募到目标位点，细菌逆转录酶产生单链供体，以及体内质粒组装。我们发现了每个系统的独特优势，并将其集成到一个名为magetic 3.0的高级系统中。我们使用magetic 3.0分析了32种环境条件下112个数量性状基因座中的因果变异，发现错义变异和多个因果变异的基因座丰富。magetic 3.0将促进基因组在单核苷酸分辨率上的功能分析，并为改进其他生物中基于模板的基因组编辑系统提供路线图。
Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.