Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum.
Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum.
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DOI：
10.3389/fbioe.2023.1213236
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发表时间：
2023
影响因子：
5.7
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中科院分区：
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产乙酸细菌可以通过将二氧化碳转化为工业相关化学品和燃料的能力，在实现净零排放方面发挥重要作用。充分利用这一潜力将依赖于有效的代谢工程工具，例如基于化脓性链球菌 CRISPR/Cas9 系统的工具。然而，将含有 cas9 的载体引入伍氏醋杆菌的尝试并未成功，这很可能是由于 Cas9 核酸酶毒性以及 cas9 基因中存在内源伍氏醋杆菌限制性修饰 (R-M) 系统的识别位点的结果。作为替代方案，本研究旨在促进 CRISPR/Cas 内源系统作为基因组工程工具的利用。因此，开发了一个 Python 脚本来自动预测原型间隔子相邻基序 (PAM) 序列，并用于识别 A. woodii I-B 型 CRISPR/Cas 系统的 PAM 候选者。分别通过干扰测定和 RT-qPCR 对鉴定出的 PAM 和天然前导序列进行了体内表征。由天然前导序列、同向重复序列和足够的间隔区组成的合成 CRISPR 阵列的表达，以及用于同源重组的编辑模板，成功地分别产生了pyrE和pheA的300bp和354bp框内删除。为了进一步验证该方法，还生成了 3.2 kb 的 hsdR1 缺失，以及 pheA 基因座上的荧光激活和吸收位移标签 (FAST) 报告基因的敲入。研究发现同源臂长度、细胞密度和用于转化的 DNA 量会显着影响编辑效率。设计的工作流程随后应用于 Clostridium autoethanogenum 的 I-B 型 CRISPR/Cas 系统，能够以 100% 的编辑效率生成 PyrE 的 561 bp 框内删除。这是首次利用 A. woodii 和 C. autoethanogenum 的内源 CRISPR/Cas 系统进行基因组工程报道。
Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO2 into industrially relevant chemicals and fuels. Full exploitation of this potential will be reliant on effective metabolic engineering tools, such as those based on the Streptococcus pyogenes CRISPR/Cas9 system. However, attempts to introduce cas9-containing vectors into Acetobacterium woodii were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous A. woodii restriction–modification (R-M) system in the cas9 gene. As an alternative, this study aims to facilitate the exploitation of CRISPR/Cas endogenous systems as genome engineering tools. Accordingly, a Python script was developed to automate the prediction of protospacer adjacent motif (PAM) sequences and used to identify PAM candidates of the A. woodii Type I-B CRISPR/Cas system. The identified PAMs and the native leader sequence were characterized in vivo by interference assay and RT-qPCR, respectively. Expression of synthetic CRISPR arrays, consisting of the native leader sequence, direct repeats, and adequate spacer, along with an editing template for homologous recombination, successfully led to the creation of 300 bp and 354 bp in-frame deletions of pyrE and pheA, respectively. To further validate the method, a 3.2 kb deletion of hsdR1 was also generated, as well as the knock-in of the fluorescence-activating and absorption-shifting tag (FAST) reporter gene at the pheA locus. Homology arm length, cell density, and the amount of DNA used for transformation were found to significantly impact editing efficiencies. The devised workflow was subsequently applied to the Type I-B CRISPR/Cas system of Clostridium autoethanogenum, enabling the generation of a 561 bp in-frame deletion of pyrE with 100% editing efficiency. This is the first report of genome engineering of both A. woodii and C. autoethanogenum using their endogenous CRISPR/Cas systems.