利用CRISPR-CAS9在酵母中构建和分析DNA序列变异文库

High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80–100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.

重点词汇

High-throughput   高通量

variant libraries   变异文库

yeast   酵母

alterations   改变

mutants   突变体

deletions   缺失

substitutions   替换

insertions   插入

en masse   全体

demonstrate   证明

utility   效用

helicase   解旋酶

highly conserved   高度保守的

residues   残基

genome-wide library 全基因组文库

open reading frames   开放阅读框

amino acids   氨基酸

scattered   分散的

vital   至关重要的

investigated   研究

precision   精确

背景知识

A genomic library is a collection of the total genomic DNA from a single organism（生物体）. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested（消化） with a restriction enzyme（限制性内切酶） to cut the DNA into fragments（片段） of a specific size. The fragments are then inserted into the vector using DNA ligase（连接酶）. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification（放大） and retrieval（回收） of specific clones（克隆） from the library for analysis.

There are several kinds of vectors available with various insert capacities（容量）. Generally, libraries made from organisms with larger genomes require vectors featuring larger inserts, thereby fewer vector molecules are needed to make the library. Researchers can choose a vector also considering the ideal insert size to find a desired number of clones necessary for full genome coverage.

Genomic libraries are commonly used for sequencing（测序） applications. They have played an important role in the whole genome sequencing of several organisms, including the human genome and several model organisms.（来源：维基百科https://en.wikipedia.org/wiki/Genomic_library）

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