Assembly and delivery of large DNA via chromosome elimination in yeast.

IF 13.1 1区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Nature Protocols Pub Date : 2025-07-10 DOI:10.1038/s41596-025-01214-z

Yuan Ma, Yao Xiong, Jiayu Xu, Hui Xu, Zongheng Fu, Guang-Rong Zhao, Yi Wu, Ying-Jin Yuan

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引用次数: 0

Abstract

Manipulation of large-scale genetic information provides a powerful approach to deciphering and engineering complex biological functions. However, the manipulation of large DNA, such as assembly and delivery, remains complex and difficult. Here we describe the experimental design strategy and protocol for a chromosome elimination-mediated large DNA assembly and delivery method (HAnDy), which enables efficient Mb-scale DNA assembly and delivery in yeast conveniently. This protocol is divided into three parts: (1) CRISPR-Cas9-mediated elimination of chromosome, which includes design and integration of a synthetic single-guide RNA (sgRNA) site near the centromere, activation of chromosome elimination by mating, and verification of the chromosome elimination. It can be used to eliminate multiple chromosomes, achieving haploidization in yeast. (2) Haploidization-mediated DNA assembly, which includes the design and construction of initial assembly strains, DNA assembly by programmed haploidization and verification of the assembled clones. (3) Haploidization-mediated DNA delivery, which includes the design and construction of inducible haploidization strains, DNA delivery by programmed haploidization and verification of the delivered clones. Users can utilize this protocol entirely or selectively depending on their needs. With the use of this protocol, it takes 10 d to achieve chromosome elimination and 7-11 d to achieve a standard cycle of haploidization-mediated DNA assembly or delivery. This protocol provides an efficient approach that is useful for the elimination, assembly and delivery of large DNA in yeast, requiring basic molecular biology skills.

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酵母中通过染色体消除的大DNA的组装和传递。

大规模遗传信息的操作为破译和工程复杂的生物功能提供了强有力的方法。然而，大型DNA的操作，如组装和传递，仍然是复杂和困难的。本文介绍了染色体消除介导的大DNA组装和传递方法（HAnDy）的实验设计策略和方案，该方法可以方便地实现酵母中mb级DNA的高效组装和传递。该方案分为三个部分：(1)crispr - cas9介导的染色体消除，包括设计和整合着丝粒附近的合成单导RNA （sgRNA）位点，通过交配激活染色体消除，验证染色体消除。它可以用来消除多个染色体，在酵母中实现单倍体化。(2)单倍体化介导的DNA组装，包括初始组装菌株的设计和构建、程序化单倍体化DNA组装和组装后克隆的验证。(3)单倍体化介导的DNA传递，包括可诱导单倍体化菌株的设计和构建、程序化单倍体化DNA传递和所传递克隆的验证。用户可以根据自己的需要完全或有选择地使用该协议。使用该方案，完成染色体消除需要10天，完成单倍体化介导的DNA组装或传递的标准周期需要7-11天。该方案提供了一种有效的方法，可用于酵母中大DNA的消除，组装和传递，需要基本的分子生物学技能。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Nature Protocols 生物-生化研究方法

CiteScore

29.10

自引率

0.70%

发文量

128

审稿时长

4 months

期刊介绍： Nature Protocols focuses on publishing protocols used to address significant biological and biomedical science research questions, including methods grounded in physics and chemistry with practical applications to biological problems. The journal caters to a primary audience of research scientists and, as such, exclusively publishes protocols with research applications. Protocols primarily aimed at influencing patient management and treatment decisions are not featured. The specific techniques covered encompass a wide range, including but not limited to: Biochemistry, Cell biology, Cell culture, Chemical modification, Computational biology, Developmental biology, Epigenomics, Genetic analysis, Genetic modification, Genomics, Imaging, Immunology, Isolation, purification, and separation, Lipidomics, Metabolomics, Microbiology, Model organisms, Nanotechnology, Neuroscience, Nucleic-acid-based molecular biology, Pharmacology, Plant biology, Protein analysis, Proteomics, Spectroscopy, Structural biology, Synthetic chemistry, Tissue culture, Toxicology, and Virology.