Roggenkamp Emily, Giersch Rachael M, Wedeman Emily, Eaton Muriel, Turnquist Emily, Schrock Madison N, Alkotami Linah, Jirakittisonthon Thitikan, Schluter-Pascua Samantha E, Bayne Gareth H, Wasko Cory, Halloran Megan, Finnigan Gregory C
Department of Biochemistry and Molecular Biophysics, Kansas State UniversityManhattan, KS, United States.
Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State UniversityManhattan, KS, United States.
Front Microbiol. 2017 Sep 20;8:1773. doi: 10.3389/fmicb.2017.01773. eCollection 2017.
continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction () and integration of desired sequences into the genome. The development of molecular toolkits and "integration cassettes" have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 "gene drive" system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome.
它继续作为基础生物学研究和工业应用的强大模型系统。全基因组范围内单独操纵菌株（文库）的发展使得高通量基因筛选成为可能，并让人们对这种单细胞真核生物有了新的全局认识。菌株构建的成功依赖于出芽酵母接受外源DNA并进行同源重组的内在能力，这使得高效质粒构建（）以及将所需序列整合到基因组中成为可能。分子工具包和“整合盒”的发展为真菌系统提供了一系列标记、删除或过表达靶基因的策略；通常，这些策略由一个C端标签（表位或荧光蛋白）、一个通用终止子序列和一个选择标记盒组成，以便于筛选。然而，使用这些传统遗传模块进行复杂菌株构建（在单个细胞中操纵多个基因组靶点）或生成全基因组文库存在后勤和技术障碍。CRISPR/Cas基因编辑技术的最新引入为包括酵母在内的许多生物系统中的多重编辑提供了一种强大的方法。我们开发了CRISPR生物技术的四种不同用途，以利用现有的、常用的酵母文库或菌株的转化来生成酵母菌株。我们提出了基于Cas9的无标记方法，用于（i）N端标记，（ii）用18种独特融合蛋白对酵母基因进行C端标记，（iii）将荧光标记菌株转化为新工程化（或密码子优化）变体，最后，（iv）使用Cas9“基因驱动”系统在二倍体菌株中快速实现亚效查询等位基因的纯合状态。这些基于CRISPR的方法证明了对先前引入基因组的靶向通用序列的应用。
