CRISPR/Cas9 RNP 介导的基因融合用于评估 Fusarium oxysporum 中的蛋白定量和亚细胞定位。

CRISPR/Cas9 RNP-Mediated Gene Fusion to Assess Protein Quantification and Subcellular Localization in Fusarium oxysporum.

机构信息

State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China.

Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA.

出版信息

Methods Mol Biol. 2022;2391:89-98. doi: 10.1007/978-1-0716-1795-3_8.

DOI:10.1007/978-1-0716-1795-3_8

PMID:34686979

Abstract

F. oxysporum is a notorious filamentous pathogenic fungus that causes serious problems in agriculture and animal/human health. Knowing how the fungus interacts throughout the course of an infection is necessary to propose an effective control strategy, and consequently the manipulation of the F. oxysporum genome is essential to investigate the molecular interplay between the host and fungus. To facilitate assessing protein quantification and subcellular localization, we developed a simple, economical CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). Reporter genes, including GFP and LacZ, can be inserted at the N- or C-terminus of an endogenous gene of interest at the original chromosomal locus, allowing partial characterization of the gene function.

摘要

尖孢镰刀菌是一种臭名昭著的丝状致病真菌，它会给农业以及动物/人类健康造成严重问题。了解真菌在整个感染过程中的相互作用对于提出有效的控制策略是必要的，因此，对尖孢镰刀菌基因组的操作对于研究宿主和真菌之间的分子相互作用至关重要。为了便于评估蛋白质定量和亚细胞定位，我们开发了一种简单、经济的基于两种不同策略的 CRISPR/Cas9 介导的内源基因标记（EGT）系统，即非同源靶向整合（HITI）和同源依赖性重组整合（HDRI）。报告基因，包括 GFP 和 LacZ，可以插入感兴趣的内源基因的 N 端或 C 端，在原始染色体位置，从而可以对基因功能进行部分表征。