Establishing a one-step marker-free CRISPR/Cas9 system for industrial Aspergillus niger using counter-selectable marker Ang-ace2.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology Letters Pub Date : 2023-12-01 Epub Date: 2023-10-08 DOI:10.1007/s10529-023-03434-3

Jiao Liu, Jie Zhu, Qian Zhang, Ruitong Lv, Hao Liu

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Abstract

Objectives: To develop a one-step, marker-free CRISPR/Cas9 system for highly efficient genome editing in industrial Aspergillus niger, with a short genetic operation cycle.

Results: Firstly, evaluation of different promoters for sgRNA expression revealed tRNA^Gly15 as the most efficient, achieving a remarkable 100% gene editing efficiency. Furthermore, a counter-selectable marker, Ang-ace2, was identified for A. niger. Subsequently, a CRISPR/Cas9 plasmid was developed, utilizing a truncated AMA1 element and the Ang-ace2 conditional expression cassette driven by a Tet-on promoter. In the presence of doxycycline, the plasmid demonstrated a 33% loss efficiency in the progeny of A. niger spores after a single generation, resulting in a shortened genetic operation cycle of 16 days for CRISPR/Cas9.

Conclusions: The one-step marker-free CRISPR/Cas9 system was successfully developed in industrial A. niger, allowing for efficient gene editing while simultaneously reducing the editing time.

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使用反选择标记Ang-ace2建立工业黑曲霉的一步无标记CRISPR/Cas9系统。

目的：开发一种一步、无标记的CRISPR/Cas9系统，用于工业黑曲霉的高效基因组编辑，遗传操作周期短。结果：首先，对sgRNA表达的不同启动子的评估显示tRNAGly15是最有效的，实现了显著的100%基因编辑效率。此外，还为黑曲霉鉴定了一种反选择性标记Ang-ace2。随后，利用截短的AMA1元件和由Tet-on启动子驱动的Ang-ace2条件表达盒，开发了CRISPR/Cas9质粒。在多西环素存在下，质粒在黑曲霉孢子单代后的子代中表现出33%的损失效率，导致CRISPR/Cas9的遗传操作周期缩短了16天。结论：在工业黑曲霉中成功开发了一步无标记的CRISPR/Cas9系统，从而允许有效的基因编辑，同时减少编辑时间。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.