crispr在大豆中的瞬时表达,简化gRNA在植物中的筛选

IF 0.7 4区 农林科学 Q3 AGRICULTURE, MULTIDISCIPLINARY
A. Koltun, Nathalia Volpi e Silva, Jéssika Angelotti-Mendonça, S. R. Marin, L. Gonçalves, A. Nepomuceno, L. M. Mertz-Henning
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引用次数: 0

摘要

本工作的目的是开发一种在大豆(Glycine max)胚胎中创建和验证CRISPR-Cas系统和不同grna的方法。两种模式基因分别用于一个gRNA的简单突变或两个向导的部分基因缺失。采用IIS型限制性内切酶将grna插入到CRISPR转化载体中,或者采用经典限制性内切酶克隆方法将启动子+ gRNA2亚克隆插入到最终的转化载体中。成功构建了1个和2个grna载体。在大豆中进行农杆菌介导的瞬时转化,以测试grna和系统本身(表达盒)的质量。经酶切富集DNA、聚合酶链反应和测序后转化的胚胎中检测到简单的突变和基因缺失,这表明CRISPR-Cas系统和向导是有效的。该协议可用于加速基于crispr的大豆遗传转化基因组编辑策略。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
CRISPR-transient expression in soybean for simplified gRNA screening in planta
Abstract The objective of this work was to develop a method to create and validate CRISPR-Cas systems and different gRNAs in soybean (Glycine max) embryos. Two model genes were used for simple mutation with one gRNA or partial gene deletion with two guides. The gRNAs were inserted into the CRISPR transformation vectors by a type IIS restriction enzyme or by subcloning and inserting the promoter + gRNA2 in the final transformation vector using the classic restriction enzyme cloning method. The vectors were successfully constructed for one and two gRNAs. Agrobacterium-mediated transient transformation in soybean was carried out to test the quality of gRNAs and of the system itself (expression cassette). Simple mutation and gene deletion were detected in the embryos transformed after DNA enrichment by enzyme digestion followed by polymerase chain reaction and sequencing, which indicates that the CRISPR-Cas system and guides were working. This protocol can be used to accelerate CRISPR-based genome editing strategies for genetic transformation in soybean.
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来源期刊
Pesquisa Agropecuaria Brasileira
Pesquisa Agropecuaria Brasileira 农林科学-农业综合
CiteScore
1.20
自引率
0.00%
发文量
45
审稿时长
9-18 weeks
期刊介绍: Pesquisa Agropecuária Brasileira – PAB – is issued monthly by Empresa Brasileira de Pesquisa Agropecuária – EMBRAPA, affiliated to Ministry of Agriculture, Livestock and Food Supply. PAB publishes original scientific-technological articles on Plant Physiology, Plant Pathology, Crop Science, Genetics, Soil Science, Food Technology and Animal Science. Its abbreviated title is Pesq. agropec. bras., and it should be used in bibliographies, footnotes, references and bibliographic strips.
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