Characterization and Engineering of a Novel Miniature Eubacterium siraeum CRISPR-Cas12f System.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2024-06-28 DOI:10.1021/acssynbio.4c00154

Yannan Wang, Yujue Wang, Na Tang, Zhipeng Wang, Deng Pan, Quanjiang Ji

引用次数: 0

Abstract

Cas12f nucleases are one of the most compact genome editors, exhibiting promising potential for in vivo therapeutic applications. However, the availability of active Cas12f genome editors remains relatively limited in the field. Here, we report the characterization and engineering of a novel miniature Cas12f endonuclease from Eubacterium siraeum (EsCas12f1, 433 amino acids). We elucidate the specific Protospacer Adjacent Motifs preference and the detailed biochemical properties for DNA targeting and cleavage. By employing rational design strategies, we systematically optimize the guide RNA of EsCas12f1, converting the initially ineffective CRISPR-EsCas12f1 system into an efficient bacterial genome editor. Furthermore, we demonstrate the capacity of EsCas12f1 for in vitro nucleic-acid diagnostics. In summary, our results enrich the miniature CRISPR-Cas toolbox and pave the way for the application of EsCas12f1 for both genome editing and in vitro diagnostics.

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一种新的微型 Eubacterium siraeum CRISPR-Cas12f 系统的特征描述和工程设计。

Cas12f 核酸酶是最紧凑的基因组编辑器之一，在体内治疗应用方面具有广阔的前景。然而，活性 Cas12f 基因组编辑器在该领域的可用性仍然相对有限。在这里，我们报告了一种新型微型Cas12f内切酶（EsCas12f1，433个氨基酸）的表征和工程设计。我们阐明了特异性原位相邻基团偏好以及 DNA 靶向和裂解的详细生化特性。通过采用合理的设计策略，我们系统地优化了 EsCas12f1 的引导 RNA，将最初无效的 CRISPR-EsCas12f1 系统转变为高效的细菌基因组编辑器。此外，我们还证明了 EsCas12f1 在体外核酸诊断方面的能力。总之，我们的研究成果丰富了微型 CRISPR-Cas 工具箱，为 EsCas12f1 在基因组编辑和体外诊断方面的应用铺平了道路。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.