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在Kluyveromyces marxianus中通过过渡的超紧CRISPR-AsCas12f1系统进行基因破坏

Kehui Zhang^1,2, Dongmei Wang^1,2, Shenglin Hu³

¹School of Life Sciences, University of Science and Technology of China, Hefei, 230027, Anhui, China.

Biotechnology letters

|July 31, 2025

概括

这项研究引入了一种新的CRISPR-AsCas12f1基因编辑方法,用于Kluyveromyces marxianus,增强微生物细胞工厂的发展. 有效的基因破坏策略改善了这种酵母物种的基因工程.

科学领域:

生物技术是生物技术.
合成生物学合成生物学
微生物工程微生物工程

背景情况:

由于其强大的生长和耐热性,Kluyveromyces marxianus是工业应用的有价值的酵母.
有效的基因破坏对于工程K. marxianus至关重要,但受到非同类重组的阻碍.
超紧的AsCas12f1CRISPR系统提供了因其小尺寸而改善基因编辑的潜力.

研究的目的:

在K. marxianus中使用AsCas12f1CRISPR系统建立一种新且高效的基因破坏方法.
通过整合tRNA-gRNA结构和破坏宿主修复基因来优化基因破坏效率.
为了证明AsCas12f1在K. marxianus.中快速生成多个基因淘汰的实用性.

主要方法:

在K. marxianus.中使用AsCas12f1的过渡基因向策略的开发.
将tRNA-gRNA数组集成到gRNA结构中,以提高编辑效率.
组合破坏宿主基因 (KmKU70,KmLIG4) 与AsCas12f1编辑以提高淘汰率.
应用优化系统,同时生成多个单基因淘汰菌株.

主要成果:

具有tRNA-gRNA集成的AsCas12f1系统显著提高了K. marxianus.中的基因破坏效率.
破坏KmKU70或KmLIG4基因进一步提高了编辑效率,接近100%.
在与KmKU70干扰相结合时,缩短的同类臂 (200 bp) 是有效的,达到87.5%的效率.
该gRNA-tRNA-array系统能够在单个转换中创建三个单基因淘汰,总效率为86.4%.

结论:

使用AsCas12f1CRISPR系统开发了一种新的,高效和快速的基因破坏策略,用于K. marxianus.
该方法通过消除构建表达矢量和删除CRISPR系统后编辑的需要来简化基因工程.
这项工作强调了酵母中AsCas12f1系统在构建微生物细胞工厂方面的广泛应用.

关键词:

克鲁伊维罗米塞斯马克西安纳斯 (Kluyveromyces marxianus) 是一个植物.这就是AsCas12f1的意思.基因破坏基因破坏这是一个gRNA-tRNA数组.Δku70 70 的时间.

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