A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences.

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Phenotypic complementation is crucial for validating gene function after knockout.
• Existing methods can be complex and may not fully confirm gene identity.

Purpose of the Study:

• To develop a streamlined and reliable method for in situ allelic replacement using CRISPR/Cas9.
• To establish a 'gold standard' for phenotypic complementation in microbial systems.

Main Methods:

• A novel strategy employing a unique 24-nucleotide 'bookmark' sequence integrated into the mutant allele.
• Utilizing the bookmark as a guide RNA target for CRISPR/Cas9-mediated replacement with a wild-type (WT) allele.
• Testing the method in *Clostridium autoethanogenum* with nine designed bookmarks.

Main Results:

• Achieved high complementation efficiencies, reaching up to 91% in *Clostridium autoethanogenum*.
• Demonstrated the successful in situ replacement of mutant alleles with WT alleles.
• Showcased the potential for incorporating 'watermark' sequences to confirm strain authenticity.

Conclusions:

• The described CRISPR/Cas9 strategy offers a simple and effective approach for 'gold standard' phenotypic complementation.
• This method enhances the reliability of gene function studies by ensuring precise allelic replacement.
• The technique is adaptable to various CRISPR/Cas systems and microbial hosts.