CRISPR/Cas9(成簇的规则间隔的短回文重复序列/CRISPR相关蛋白9)已成为在作物物种中引发靶向遗传改变的首选育种工具,作为改善多种农艺性状的手段,包括抗病性,近年来。随着近年来CRISPR/Cas9技术在紫花苜蓿(苜蓿)中的应用,这是一种重要的多年生牧草豆类,它用于增强病原体抗性的用途几乎肯定在地平线上。在这一章中,我们提供了通过CRISPR/Cas9在苜蓿的精确基因组位点产生单个非同源末端连接衍生的indel的详细程序。此方法包括此过程中的关键步骤,包括引导RNA设计,二进制CRISPR载体构建,农杆菌介导的苜蓿外植体转化,以及转化基因型的分子评估,用于转基因和编辑鉴定。
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has become a breeding tool of choice for eliciting targeted genetic alterations in crop species as a means of improving a wide range of agronomic traits, including disease resistance, in recent years. With the recent development of CRISPR/Cas9 technology in Medicago sativa (alfalfa), which is an important perennial forage legume grown worldwide, its use for the enhancement of pathogen resistance is almost certainly on the horizon. In this chapter, we present detailed procedures for the generation of a single nonhomologous end-joining-derived indel at a precise genomic locus of alfalfa via CRISPR/Cas9. This method encompasses crucial steps in this process, including guide RNA design, binary CRISPR vector construction, Agrobacterium-mediated transformation of alfalfa explants, and molecular assessments of transformed genotypes for transgene and edit identification.