利用融合了 UGI 降解标签的目标 AID 系统编辑链霉菌基因组

IF 3.9 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Engineering in Life Sciences Pub Date : 2024-06-24 DOI:10.1002/elsc.202400005
Pamella Apriliana, Prihardi Kahar, Norimasa Kashiwagi, Akihiko Kondo, Chiaki Ogino
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引用次数: 0

摘要

利用链霉菌作为开发创新药物和药用化合物的微生物底盘,展示了其生产生物活性天然物质的能力。最近对成簇规律性间隔短回文重复(CRISPR)技术的关注凸显了其在基因组编辑方面的潜力。然而,在某些微生物菌株(尤其是链霉菌)中应用 CRISPR 技术会遇到一些特定的挑战。这些挑战包括实现高效的基因表达和保持遗传稳定性,这对成功进行基因组编辑至关重要。为了克服这些障碍,我们开发了一种创新方法,该方法结合了几个关键要素:活化诱导胞苷脱氨酶(AID)、核酸酶缺陷cas9变体(dCas9)和Petromyzon marinus胞苷脱氨酶1(PmCDA1)。在本研究中,我们采用了这一新策略来改造一种链霉菌(Streptomyces coelicolor)菌株。目标基因是 actVA-ORF4 (SCO5079),它参与放线菌素的生产。工程过程包括引入一个特定的构建体[pGM1190-dcas9-pmCDA-UGI-AAV-actVA-ORF4 (SCO5079)]来创建一个 CrA10 突变菌株。产生的 CrA10 突变株不产生放线菌素。这一结果凸显了这种组合方法在链霉菌遗传操作方面的潜力。CrA10 突变体未能产生放线菌素,这最终证明在目标位点的基因编辑是成功的,从而肯定了这种方法在链霉菌中进行精确基因修饰的有效性。
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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Editing Streptomyces genome using target AID system fused with UGI-degradation tag

The utilization of Streptomyces as a microbial chassis for developing innovative drugs and medicinal compounds showcases its capability to produce bioactive natural substances. Recent focus on the clustered regularly interspaced short palindromic repeat (CRISPR) technology highlights its potential in genome editing. However, applying CRISPR technology in certain microbial strains, particularly Streptomyces, encounters specific challenges. These challenges include achieving efficient gene expression and maintaining genetic stability, which are critical for successful genome editing. To overcome these obstacles, an innovative approach has been developed that combines several key elements: activation-induced cytidine deaminase (AID), nuclease-deficient cas9 variants (dCas9), and Petromyzon marinus cytidine deaminase 1 (PmCDA1). In this study, this novel strategy was employed to engineer a Streptomyces coelicolor strain. The target gene was actVA-ORF4 (SCO5079), which is involved in actinorhodin production. The engineering process involved introducing a specific construct [pGM1190-dcas9-pmCDA-UGI-AAV-actVA-ORF4 (SCO5079)] to create a CrA10 mutant strain. The resulting CrA10 mutant strain did not produce actinorhodin. This outcome highlights the potential of this combined approach in the genetic manipulation of Streptomyces. The failure of the CrA10 mutant to produce actinorhodin conclusively demonstrates the success of gene editing at the targeted site, affirming the effectiveness of this method for precise genetic modifications in Streptomyces.

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来源期刊
Engineering in Life Sciences
Engineering in Life Sciences 工程技术-生物工程与应用微生物
CiteScore
6.40
自引率
3.70%
发文量
81
审稿时长
3 months
期刊介绍: Engineering in Life Sciences (ELS) focuses on engineering principles and innovations in life sciences and biotechnology. Life sciences and biotechnology covered in ELS encompass the use of biomolecules (e.g. proteins/enzymes), cells (microbial, plant and mammalian origins) and biomaterials for biosynthesis, biotransformation, cell-based treatment and bio-based solutions in industrial and pharmaceutical biotechnologies as well as in biomedicine. ELS especially aims to promote interdisciplinary collaborations among biologists, biotechnologists and engineers for quantitative understanding and holistic engineering (design-built-test) of biological parts and processes in the different application areas.
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