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开发一种基于Saccharopolyspora spinosa内源型I-B CRISPR-Cas系统的强大的基因组编辑工具

Wenfang Wang¹, Huiyan He¹, Hewei Liu¹

¹College of Life Sciences, Shanghai Normal University, Shanghai, 200234, China.

Science China. Life sciences

|March 25, 2025

概括

研究人员开发了一种新的CRISPR-Cas基因组编辑工具,用于Saccharopolyspora spinosa,从而实现高效的基因删除和插入. 这种工具克服了遗传障碍,促进了这种工业性菌体的生物杀虫剂生产工程.

科学领域:

微生物学微生物学
生物技术是生物技术.
遗传学是一个遗传学.

背景情况:

螺旋菌生产有价值的螺旋生物杀虫剂.
由于有限的工具和遗传不可访问性,对S. spinosa的基因操纵具有挑战性.

研究的目的:

在S. spinosa.中描述一种内源型I-BCRISPR-Cas系统.
为S. spinosa.开发高效的基因组工程工具.
克服限制-修改 (RM) 系统障碍,以提高转型.

主要方法:

生物信息学分析和等离子体干扰测试用于描述CRISPR-Cas系统及其PAMs.
用miniCRISPR阵列和修复模板设计和交付编辑等离子体.
对I型和II型限制修改系统的系统性破坏.
删除75kb的spinosyn生物合成基因集群 (BGC) 和基因插入.

主要成果:

使用开发的CRISPR-Cas工具实现了100%的编辑效率,用于删除基因.
三个RM系统同时解除武装,使等离子体转换效率提高了约3.9倍 (复制性) 和约4.2倍 (整合性).
在工程菌株中实现了有效的spinosyn BGC删除和高效基因插入.

结论:

基于内源性I型CRISPR-Cas系统的可靠和高效的基因组编辑工具已为S. spinosa.建立.
本文介绍了第一个基于CRISPR-Cas的内源基因组编辑系统,用于非模型的工业菌体.
开发的工具和RM系统裁军战略显著提高了S. spinosa的遗传处理能力.

关键词:

糖多胞体是什么糖多胞体是什么糖多胞体是什么糖多胞体是什么内生类型I-B的CRISPR-Cas系统.基因组工程是基因组工程.限制修改 (RM) 系统

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