Genome-scale CRISPRi and base-editing libraries for genetic decoding and strain engineering in Shewanella

Area of Science:

• Microbiology
• Synthetic Biology
• Genomics

Background:

• Understanding microbial metabolic networks is crucial, especially in non-model organisms.
• CRISPR-based tools offer powerful gene-editing functions like knockdown and mutation.

Purpose of the Study:

• To design and validate CRISPR-based libraries for the electroactive Shewanella oneidensis MR-1.
• To enable deeper genomic interrogation and expand the application of this microorganism.

Main Methods:

• Designed three CRISPR-based libraries: CRISPR interference (CRISPRi), protein mutation, and inactivation libraries.
• Developed a conjugation-based transformation method for high-coverage library delivery.
• Utilized single-guide RNAs (sgRNAs) for precise gene targeting.

Main Results:

• Created comprehensive CRISPR libraries targeting 99.6% of S. oneidensis MR-1 genes.
• Validated library design, construction, and transformation methods.
• Identified potential essential genes and expanded the substrate range for electricity generation (glucose, chitin).

Conclusions:

• Genome-scale CRISPR tools are effective for undercharacterized microorganisms like Shewanella.
• This work provides a framework for applying similar tools to other microbial species.
• Expanded S. oneidensis MR-1's utility in bioelectrochemical applications.