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容易固化和pH调节的CRISPR-Cas9等离子体用于Lactococcus cremoris中的基因编辑和等离子体固化

Javier Nicolás Garay-Novillo^1,2, José Ángel Ruiz-Masó², Gloria Del Solar²

¹Departamento de Química Biológica Ranwel Caputto, CIQUIBIC-CONICET, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Microbial biotechnology

|December 21, 2024

概括

我们开发了一种CRISPR-Cas9系统,用于编辑乳球球菌,从而实现高效的等离子体治愈和染色体修改. 这种多功能工具有助于生成基因编辑菌株,而无需Cas9毒性或需要选择性压力.

科学领域:

微生物学微生物学
分子生物学分子生物学
基因工程是一种基因工程.

背景情况:

乳球球菌是重要的乳制品初始培养物,但基因操纵可能具有挑战性.
现有的L. cremoris遗传修饰方法往往需要选择性压力,并且可能是低效的.

研究的目的:

开发一种基于CRISPR-Cas9的多功能和高效的基因编辑系统,用于Lactococcus cremoris.
为了使等离子体和染色体编辑,包括无等离子体菌株的生成.

主要方法:

在可诱导和构成性促进体下构建表达Cas9核酶和单导向RNA的穿载体.
使用pH调节的促进剂来调节Cas9表达,并使用sgRNA强大的构成性促进剂.
实施了一个主体独立的重组系统 (λRed-phage Redβ重组酶和 EcSSB) 以增强染色体编辑.

主要成果:

在L. cremoris.中证明了低和高复制量等离子体的高效等离子体治愈.
通过调节Cas9表达来实现可调节的准效率.
通过使用CRISPR-Cas9结合内源性或Redβ-EcSSB辅助重组,成功生成了数千个基因编辑细胞.
在没有选择性压力的情况下,没有观察到与Cas9相关的毒性和高载体损失率.

结论:

开发的CRISPR-Cas9系统为L. cremoris.的基因工程提供了一个强大而灵活的工具.
这一策略简化了精确修饰的,无等离子体的L. cremoris菌株的生成.
该系统的效率,捕鱼性和缺乏毒性使其对乳制品初始培养的发展非常有价值.

关键词:

乳球球菌克雷莫里斯 (英语:Lactococcus cremoris) 是一种乳球球菌.克里斯普尔-卡斯9是什么意思基因编辑是一种基因编辑.塑固化固化等离子体塑的稳定性塑的稳定性

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