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<p>To prevent the unique difficulty of hygromycin-based gene editing on industrial <i>A. niger</i> strain and increase the working efficiency, the local <i>pyrG</i> marker was removed by well-designed dual sgRNAs and repair template through Cas9-ribonucleoprotein (RNP) strategy in this study. The positive rate of the desired <i>pyrG</i> auxotroph construction was 100%, while no transformant was observed using the traditional methods. The complementation strain showed similar fermentation character as the starting strain. Moreover, an efficient and seamless knock out plasmid-based strategy was established, achieving positive rate at 90% and 50% for challenging Δ<i>ku70</i> and Δ<i>ku80</i> respectively. Further, combined hygromycin markers and miniaturization cultivation were conducted to select the poor growth strain. Finally, skillfully designed sgRNA and <i>amdS</i> counter-selection repair template were used to obtain <i>ERG3</i><sup>Tyr185</sup> mutation. A highly-efficient precise strategy was established for <i>A. niger</i> through a diagnostic PCR method, with nearly 100% positive rate. Highly- precise desired point mutation was achieved by the developed gene toolbox.</p>

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Constructing pyrG marker by CRISPR/Cas facilities the highly-efficient precise genome editing on industrial Aspergillus niger strain

  • 期刊论文

  • 发表日期：

    2025年2月16日

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  • Jingchun Sun,
  • Xing Jiang,
  • Feng Xu,
  • Xiwei Tian,
  • Ju Chu

摘要

To prevent the unique difficulty of hygromycin-based gene editing on industrial A. niger strain and increase the working efficiency, the local pyrG marker was removed by well-designed dual sgRNAs and repair template through Cas9-ribonucleoprotein (RNP) strategy in this study. The positive rate of the desired pyrG auxotroph construction was 100%, while no transformant was observed using the traditional methods. The complementation strain showed similar fermentation character as the starting strain. Moreover, an efficient and seamless knock out plasmid-based strategy was established, achieving positive rate at 90% and 50% for challenging Δku70 and Δku80 respectively. Further, combined hygromycin markers and miniaturization cultivation were conducted to select the poor growth strain. Finally, skillfully designed sgRNA and amdS counter-selection repair template were used to obtain ERG3Tyr185 mutation. A highly-efficient precise strategy was established for A. niger through a diagnostic PCR method, with nearly 100% positive rate. Highly- precise desired point mutation was achieved by the developed gene toolbox.