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在酵母中使用CRISPR/Cas9和高活性cytidine和adeninedeaminases进行向的活体变

Christos Skrekas¹, Angelo Limeta¹, Verena Siewers^1,2

¹Department of Life Sciences, Chalmers University of Technology, Gothenburg SE-41296, Sweden.

ACS synthetic biology

|July 24, 2023

概括

这项研究提出了一种新的CRISPR-Cas9工具包,用于酵母中的向DNA突变,增强蛋白质工程. 开发的系统为定向进化提供了高效率和控制,克服了传统方法的局限性.

科学领域:

分子生物学分子生物学
生物技术是生物技术.
合成生物学合成生物学

背景情况:

定向进化对于改善蛋白质功能至关重要,特别是代谢途径中的酶.
目前的方法,如易出错的PCR,受到图书馆大小和细胞转换效率的限制.
向体内突变发生提供了一个有希望的替代方案来克服这些局限性.

研究的目的:

开发一种高效率,时间可控的Saccharomyces cerevisiae的向突变发生工具包.
利用CRISPR/Cas9技术进行精确的体内DNA编辑.
通过扩大变体库生成来增强定向进化策略.

主要方法:

dCas9蛋白与过度活跃的腺因和细胞因子除氨酶的融合.
开发一种可诱导的基于CRISPR的突变发生器工具,用于特定的DNA向.
研究指导RNA复杂化对突变发生效率的影响.

主要成果:

在体内实现了高的突变发生效率和扩展的编辑窗口.
证明了对DNA突变发生的时间可控和有针对性的方法.
量化了指导RNA复合对表型和DNA水平突变发生的影响.

结论:

开发的CRISPR/Cas9工具包提供了一种有效和可控制的方法,用于酵母中的向突变发生.
这种工具通过允许更大,更多样化的蛋白质变体库来推进定向进化.
这些发现有助于为生物技术应用改进功能的蛋白质的工程.

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