CRISPR/Cas9-Based Genome Editing of the Saccharomyces cerevisiae ADE2 Gene with Restriction-Free Cloning and a Rapid BamHI Digest Readout

Current Protocols Essential Laboratory Techniques Pub Date : 2020-12-09 DOI:10.1002/cpet.45

Allison R. Sirois, Nils Pilotte, Steven A. Williams, Lori J. Saunders

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (CRISPR/Cas) are revolutionary tools for predictable and repeatable genome editing. In this article, the CRISPR/Cas9 system will be used to engineer the genome of the yeast Saccharomyces cerevisiae. As a model organism utilized across biological disciplines, efficient and tailorable methods for engineering the yeast genome are indispensable for a variety of research, educational, and commercial applications. The protocols described here incorporate a simple restriction-free cloning strategy and digestion-based screening method that can be readily and easily modified to suit user-designed experimental needs. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Recombinant pCAS plasmid preparation

Basic Protocol 2: Barcode/editing fragment assembly

Basic Protocol 3: Gene editing by yeast co-transformation

Alternate Protocol: Competent yeast preparation and transformation by a lithium acetate/single-stranded carrier method

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基于CRISPR/Cas9的酿酒酵母ADE2基因的基因组编辑，无限制性克隆和快速BamHI文摘读取

聚集规律间隔短回文重复序列(CRISPR)和CRISPR相关系统(CRISPR/Cas)是可预测和可重复的基因组编辑的革命性工具。在本文中，CRISPR/Cas9系统将用于酵母的基因组工程。作为一种跨生物学科使用的模式生物，高效和可定制的酵母基因组工程方法对于各种研究，教育和商业应用是必不可少的。这里描述的方案包括一个简单的无限制克隆策略和基于消化的筛选方法，可以很容易地修改，以适应用户设计的实验需要。©2020 Wiley期刊有限责任公司

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