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The pivotal role of phyto-microbiome as a decisive factor of plant productivity and well-being is irrefutable. The members of the phyto-microbiome are diverse and they co-exist forming complex interactions with the plant. Within the phyto-microbiome, the Bacillus subtilis CF3 strain is widely known for alleviating stress. By editing its genome, it can further be engineered to produce two enzymes, IaaM and IaaH, which can metabolize several intermediates to produce IAA, a hormone crucial for the regulation of developmental processes in the plants. The two genes encoding for IaaM and IaaH are introduced into the B. subtilis CF3 strain by using an optimized CRISPR/Cas9 system employing a two-step strategy, using an Antibiotic Resistance Cassette as a selection marker. This method can be made efficient in genome editing since it employs different plasmids carrying CRISPR tools along with a separate plasmid carrying the donor DNA to achieve the insertion without any undesired mutations. Thereby, this chapter confers a protocol for editing the Genome of B. subtilis CF3 recruiting this novel optimized CRISPR CAS system.

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Bacillus Genome Engineering Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

  • 章节

  • 发表日期：

    2025年

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  • Mullainathan Muthukumaran,
  • Ida Nancy K.,
  • Sanjay B. Vasan,
  • Dhanasekaran Dharumadurai

摘要

The pivotal role of phyto-microbiome as a decisive factor of plant productivity and well-being is irrefutable. The members of the phyto-microbiome are diverse and they co-exist forming complex interactions with the plant. Within the phyto-microbiome, the Bacillus subtilis CF3 strain is widely known for alleviating stress. By editing its genome, it can further be engineered to produce two enzymes, IaaM and IaaH, which can metabolize several intermediates to produce IAA, a hormone crucial for the regulation of developmental processes in the plants. The two genes encoding for IaaM and IaaH are introduced into the B. subtilis CF3 strain by using an optimized CRISPR/Cas9 system employing a two-step strategy, using an Antibiotic Resistance Cassette as a selection marker. This method can be made efficient in genome editing since it employs different plasmids carrying CRISPR tools along with a separate plasmid carrying the donor DNA to achieve the insertion without any undesired mutations. Thereby, this chapter confers a protocol for editing the Genome of B. subtilis CF3 recruiting this novel optimized CRISPR CAS system.