An Efficient Targeted Mutagenesis System Using CRISPR/Cas in Monocotyledons

Q1 Agricultural and Biological Sciences

Current protocols in plant biology Pub Date : 2016-06-10 DOI:10.1002/cppb.20021

Zhen Liang, Yuan Zong, Caixia Gao

引用次数: 10

Abstract

Precise genome modification using artificial nucleases is a powerful tool for in-depth understanding of gene functions and for creating new varieties. The CRISPR/Cas system, derived from an adaptive immunity system in bacteria and archaea, can introduce DNA double-strand breaks (DSBs) into pre-selected genomic loci and lead to loss of gene function due to error-prone non-homologous end joining (NHEJ). RNA-guided nucleases have been widely used in several eukaryotic organisms. In this article, we provide a detailed protocol for designing and constructing gRNA targets, detecting nuclease activity in transient protoplast assays, and identifying mutations in transgenic plants (including rice, wheat and maize). Targeted mutations in T0 plants can be generated in 14 to 18 weeks. © 2016 by John Wiley & Sons, Inc.

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基于CRISPR/Cas的单子叶高效靶向诱变系统

利用人工核酸酶进行精确的基因组修饰是深入了解基因功能和创造新品种的有力工具。CRISPR/Cas系统源于细菌和古细菌的适应性免疫系统，可以将DNA双链断裂(DSBs)引入预先选择的基因组位点，并由于易出错的非同源末端连接(NHEJ)导致基因功能丧失。rna引导核酸酶已广泛应用于几种真核生物中。在本文中，我们提供了一个详细的方案来设计和构建gRNA靶点，检测瞬时原生质体的核酸酶活性，以及鉴定转基因植物(包括水稻、小麦和玉米)的突变。T0植株的靶向突变可在14 - 18周内产生。©2016 by John Wiley &儿子,Inc。

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

Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.