一种将多个 gRNA 组装到直接载体中进行番茄基因组编辑的综合方案。

A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato.

机构信息

Repository of Tomato Genomics Resources, Department of Plant Sciences, University of Hyderabad, Hyderabad, India.

ATGC Biotech Pvt. Ltd, Hyderabad, India.

出版信息

Methods Mol Biol. 2024;2788:317-335. doi: 10.1007/978-1-0716-3782-1_19.

DOI:10.1007/978-1-0716-3782-1_19

PMID:38656523

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.

摘要

簇状规律间隔短回文重复序列 (CRISPR)-Cas9（CRISPR 相关蛋白 9）是一种强大的 DNA 编码、RNA 介导的序列特异性核酸酶系统，广泛用于各种植物的基因组编辑。尽管有许多关于 gRNA 组装和植物转化的报道，但番茄中完整的基因编辑方法还没有一个单一的资源。本章提供了一个全面的方案，用于设计 gRNA、将其组装到载体中、进行植物转化以及最终在番茄中进行突变体分析。