Multiplexed Genome Editing and Transcriptional Knockdown in Yarrowia lipolytica by CRISPR-Cpf1 and an Orthogonal T7 System

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-08-14 DOI:10.1021/acssynbio.5c00104

Hanqing Zhang, Luai R. Khoury and Peng Xu*,

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引用次数: 0

Abstract

Yarrowia lipolytica, a nonconventional yeast, has become an industrial workhorse to synthesize valuable compounds, including lipids, oleochemicals, and nutraceuticals. While the synthetic biology toolkits to engineer the genome and endogenous metabolic pathways are not as developed as Baker’s yeast, it has emerged as a promising microbial host for industrial applications. In this study, we examined the multiplexed editing capability of the CRISPR-AsCpf1 coupled with gRNAs generated from either a yeast native promoter or an orthogonal T7 promoter, which yielded 73.3% editing efficiency for up to four target genes and 100% editing efficiency for two genes. We also attempted two strategies to enhance homology-directed recombination (HDR) efficiency; only minor improvements were observed. We further demonstrated that CRISPR-dAsCpf1 with T7-driven gRNA achieved significant gene knockdown compared to the CRISPR-RfxCas13d system. Its knockdown efficiency was comparable to that of an antisense T7 promoter system. Taken together, this work provides a facile toolkit that enables efficient and multiplexed genome editing and transcriptional knockdown of critical genes by combining CRISPR-Cpf1 with an orthogonal T7 transcription system in Y. lipolytica.

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利用CRISPR-Cpf1和正交T7系统多重基因组编辑和转录敲低脂耶氏菌。

脂溶耶氏酵母是一种非传统的酵母，它已经成为工业上合成有价值的化合物的主要原料，包括脂质、油脂化学物质和营养药品。虽然用于基因工程和内源性代谢途径的合成生物学工具包不如贝克酵母发达，但它已成为工业应用中有前途的微生物宿主。在这项研究中，我们检测了CRISPR-AsCpf1与酵母原生启动子或正交T7启动子产生的grna偶联的多重编辑能力，对多达四个目标基因的编辑效率为73.3%，对两个基因的编辑效率为100%。我们还尝试了两种策略来提高同源定向重组（HDR）的效率；只观察到微小的改善。我们进一步证明，与CRISPR-RfxCas13d系统相比，带有t7驱动gRNA的CRISPR-dAsCpf1实现了显著的基因敲低。其敲除效率与反义T7启动子系统相当。综上所述，这项工作提供了一个简单的工具包，通过将CRISPR-Cpf1与脂肪瘤的正交T7转录系统相结合，实现高效和多重的基因组编辑和关键基因的转录敲低。

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.