CRISPR/Cas9在高粱靶向诱变中的应用

Q1 Agricultural and Biological Sciences

Current protocols in plant biology Pub Date : 2020-06-05 DOI:10.1002/cppb.20112

Si Nian Char, Hyeyoung Lee, Bing Yang

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引用次数: 9

摘要

高粱(高粱双色)满足了对生物能源的需求，也为世界人口提供了大量的膳食热量。因此，许多生物学研究都将高粱作为驯化粮食作物和生物能源作物改良的研究模型。此外，在大量禾本科植物物种中利用基因组编辑系统已经得到了广泛的研究，高粱也不例外。然而，一项详细介绍使用CRISPR/Cas9的基因组编辑策略的协议尚未被报道，该协议结合了基于农杆菌介导的DNA转移的高粱高效组织培养和转化平台。该方案概述了以BTx623为参考基因组设计高粱CRISPR靶点、构建高粱CRISPR/Cas9质粒、组织培养、农杆菌介导的转化以及CRISPR/Cas9诱导突变体的基因分型的步骤和工作流程。©2020 Wiley期刊有限公司基本协议1:构建CRISPR/Cas9表达载体分析CRISPR编辑植物基本协议2:高粱的稳定转化支持协议:高粱植物在温室中的管理

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Use of CRISPR/Cas9 for Targeted Mutagenesis in Sorghum

Sorghum (Sorghum bicolor) fulfills the demand for bioenergy resources and also provides substantial diet calories to the world's population. Therefore, many biological studies use sorghum as a research model for improvement of the domesticated food and bioenergy crops. Furthermore, leveraging genome editing systems in a plethora of grass plant species has been extensively studied with no exception in sorghum. However, a protocol that details the genome editing strategies using CRISPR/Cas9 and that combines an efficient tissue culture and transformation platform in sorghum based on Agrobacterium-mediated DNA transfer has yet to be reported. This protocol outlines the steps and workflow from design of sorghum CRISPR target sites using BTx623 as a reference genome, construction of sorghum CRISPR/Cas9 plasmids, tissue culture, to Agrobacterium-mediated transformation followed by genotyping of CRISPR/Cas9 induced mutants. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Construction of CRISPR/Cas9 expression vector to analysis of CRISPR-edited plants

Basic Protocol 2: Stable transformation of sorghum

Support Protocol: Management of sorghum plants in a greenhouse

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来源期刊

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

自引率

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发文量

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.