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一个Cas3基编辑工具,用于可向的体内突变发生

Anna Zimmermann^1,2, Julian E Prieto-Vivas^1,2, Charlotte Cautereels^1,2

¹VIB Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, Leuven, 3001, Belgium.

Nature communications

|June 9, 2023

概括

CoMuTER (使用I-E型CRISPR-Cas系统进行受限突变) 能够为蛋白质工程提供有针对性的,大规模的DNA突变. 这种工具成功地将酵母中的利科产量翻了一番,证明了其在路径优化中的实用性.

科学领域:

合成生物学合成生物学
分子生物学分子生物学
生物技术是生物技术.

背景情况:

遗传多样性对于蛋白质工程和代谢途径优化至关重要.
现有的随机突变发生法方法在针对特定的基因组区域方面存在局限性.
需要能够实现精确且大规模的体内DNA突变发生的工具.

研究的目的:

开发一种用于诱导和向大型基因组位点的体内可诱导和可向的突变发生的新工具.
通过受限突变发生,使代谢途径的高效工程成为可能.
克服当前全基因组或狭窄窗口突变发生的方法的局限性.

主要方法:

开发了CoMuTER (使用I-E型CRISPR-Cas系统进行受限突变).
使用Cas3螺旋酶与细胞因子脱氨酶融合,以解和突变大片DNA.
应用CoMuTER用于高达55千基基因组位点的诱导性和向性突变发生.

主要成果:

与基因组的其他部分相比,CoMuTER在目标区域内的突变频率增加了350倍.
在目标位点中观察到平均每千基基因0.3个突变的突变率.
经过一轮突变发生后,通过在Saccharomyces cerevisiae中翻倍烯生产来证明成功的途径优化.

结论:

CoMuTER 是一种有效的工具,可以在大型基因组区域中产生有针对性的遗传多样性.
该系统促进了高效的代谢通路工程和蛋白质工程应用.
CoMuTER显著提升了体内突变和菌株开发的能力.

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