Species- and site-specific genome editing in complex bacterial communities

Area of Science:

• Microbiology
• Genetics
• Bioinformatics

Background:

• Studying microbial gene functions traditionally requires culturing microorganisms, but most microbes remain uncultured.
• This limitation hinders genetic analysis and understanding of interactions in uncultured microbial communities.
• A need exists for methods to genetically manipulate specific organisms within complex microbial consortia.

Purpose of the Study:

• To develop and validate a generalizable strategy for editing genomes of specific organisms within microbial communities.
• To enable targeted genetic manipulation for understanding gene functions and controlling microbial populations.
• To overcome the limitations of traditional methods for studying uncultured microbes.

Main Methods:

• Environmental transformation sequencing (ET-seq) was used to identify genetically tractable bacteria and archaea within a community by mapping transposon insertions.
• DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART) systems were employed for targeted DNA insertion into identified tractable organisms.
• The combined ET-seq and DART approach was applied to soil and infant gut microbiota samples.

Main Results:

• Successful species- and site-specific genome editing was achieved in several bacterial species within both soil and infant gut microbial communities.
• Gene fitness was measured in a nonmodel bacterium, demonstrating the utility of the method for functional genomics.
• The targeted species were successfully enriched, showcasing the potential for microbial community control.

Conclusions:

• The developed ET-seq and DART tools provide a powerful and generalizable strategy for genetic manipulation of specific microbes within their natural communities.
• These methods significantly advance the ability to study gene functions and interactions of previously uncultured microorganisms.
• The tools offer new possibilities for understanding and engineering microbial communities for various applications.