UltraCAST: A Flexible All-In-One Suicide Vector for Modifying Bacterial Genomes Using a CRISPR-Associated Transposon.

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• CRISPR-associated transposons (CASTs) are mobile genetic elements guided by RNA, commonly found in bacterial genomes.
• Existing genome editing tools often lack efficiency or applicability in non-model bacterial species.

Purpose of the Study:

• To develop and demonstrate a novel genome editing tool, UltraCAST, for precise DNA insertion in bacteria.
• To showcase the utility of UltraCAST in a non-model bacterial species, *Serratia symbiotica*.

Main Methods:

• Engineered a suicide vector, UltraCAST, incorporating the *Vibrio cholerae* Type I-F CAST system.
• Integrated Golden Gate assembly sites for guide RNA and mini-transposon cargo cloning.
• Utilized fluorescent protein gene dropouts for selection and validation.

Main Results:

• Successfully demonstrated UltraCAST-mediated genome editing by disrupting a specific gene in *Serratia symbiotica* CWBI-2.3 ^T.
• Validated the flexibility of UltraCAST for inserting DNA into targeted genomic locations.
• Confirmed the system's functionality in a non-model organism lacking established genetic tools.

Conclusions:

• UltraCAST provides a versatile and efficient platform for bacterial genome engineering.
• This tool expands the possibilities for genetic manipulation in a broader range of bacterial species.
• Facilitates the study of gene function and genome editing in previously challenging bacterial systems.