CILF: CRISPR/Cas9 based integration of large DNA fragments in Saccharomyces cerevisiae

IF 3.5 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology and Bioengineering Pub Date : 2024-08-18 DOI:10.1002/bit.28830

Shijie Xu, Jie Meng, Qi Zhang, Baisong Tong, Zihe Liu, Jinyu Fu, Shuobo Shi

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Abstract

Genome integration technology has markedly expedited the construction of cell factories. However, its application is currently limited by the inefficient integration of large DNA fragments. Here, we report a CRISPR/Cas9 based integration of large DNA fragments (CILF) method to efficiently integrate large DNA fragments in Saccharomyces cerevisiae. In this approach, a fusion protein, Cas9-Brex27-FadR, was employed for the targeted delivery of donor plasmid to double-strand breaks (DSBs), while simultaneously recruiting Rad51 to enhance the efficiency of homologous recombination (HR). Our findings demonstrate that this method can achieve an integration efficiency of 98% for 10 kb DNA fragments and nearly 80% for 40 kb DNA fragments at a single site, using donor plasmids with 1000 bp homology arms (HAs) and 12 FadR binding sites (BSs). The CILF technique significantly enriches the synthetic biology toolbox of S. cerevisiae, offering significant potential to propel advancements in both synthetic biology and metabolic engineering.

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CILF：基于 CRISPR/Cas9 技术在酿酒酵母中整合大 DNA 片段。

基因组整合技术大大加快了细胞工厂的建造速度。然而，由于大 DNA 片段的整合效率不高，该技术的应用目前受到限制。在这里，我们报告了一种基于CRISPR/Cas9的大DNA片段整合（CILF）方法，可在酿酒酵母中高效整合大DNA片段。在这种方法中，Cas9-Brex27-FadR融合蛋白被用于将供体质粒定向传递到双链断裂（DSB）处，同时招募Rad51以提高同源重组（HR）的效率。我们的研究结果表明，使用具有1000 bp同源臂（HA）和12个FadR结合位点（BS）的供体质粒，这种方法在单个位点对10 kb DNA片段的整合效率可达98%，对40 kb DNA片段的整合效率接近80%。CILF 技术极大地丰富了 S. cerevisiae 的合成生物学工具箱，为推动合成生物学和代谢工程的发展提供了巨大潜力。

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来源期刊

Biotechnology and Bioengineering 工程技术-生物工程与应用微生物

CiteScore

7.90

自引率

5.30%

发文量

280

审稿时长

2.1 months

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