Presentation Information
[3GET-13-KL]Functional gene insertion using a type I-F3 CRISPR-associated transposon
○Tomoyuki Numata1, Kazuki Ishihara1 (1. Kyushu University (Japan))
Keywords:
CRISPR-associated transposon,I-F3 CAST
CRISPR-associated transposons (CASTs) are promising tools for site-specific DNA integration because they enable programmable DNA insertion without requiring double-strand break repair. These systems rely on RNA-guided target recognition by CRISPR-Cas effectors and the coordinated action of transposition-associated proteins. Although their potential for genome engineering has attracted increasing attention, the molecular basis by which accurate target recognition is coupled to productive DNA transposition remains incompletely understood.
Here, we describe the genome editing potential of the type I-F3 CAST from Vibrio parahaemolyticus (VpCAST) and summarize key mechanistic insights obtained from structural and functional analyses. Our studies revealed how VpCAST recognizes target DNA and showed that correct guide-target pairing in the PAM-distal region is important for efficient transposition. These findings provide a mechanistic basis for understanding how CAST systems achieve accurate RNA-guided DNA insertion.
We also demonstrate the application of VpCAST to functional gene insertion in the Escherichia coli genome. Using the methionine auxotrophic strain B834(DE3), which carries a deletion within the metE gene, we tested whether VpCAST could restore gene function through targeted DNA integration. A crRNA was designed to direct insertion near the native metE promoter, and an intact metE gene was successfully integrated into the genome. This insertion restored growth under methionine-depleted conditions, demonstrating functional complementation. PCR and Sanger sequencing confirmed accurate genomic integration of the donor DNA.
Together, these results highlight the potential of VpCAST as a programmable platform for functional gene knock-in and bacterial genome editing.
Here, we describe the genome editing potential of the type I-F3 CAST from Vibrio parahaemolyticus (VpCAST) and summarize key mechanistic insights obtained from structural and functional analyses. Our studies revealed how VpCAST recognizes target DNA and showed that correct guide-target pairing in the PAM-distal region is important for efficient transposition. These findings provide a mechanistic basis for understanding how CAST systems achieve accurate RNA-guided DNA insertion.
We also demonstrate the application of VpCAST to functional gene insertion in the Escherichia coli genome. Using the methionine auxotrophic strain B834(DE3), which carries a deletion within the metE gene, we tested whether VpCAST could restore gene function through targeted DNA integration. A crRNA was designed to direct insertion near the native metE promoter, and an intact metE gene was successfully integrated into the genome. This insertion restored growth under methionine-depleted conditions, demonstrating functional complementation. PCR and Sanger sequencing confirmed accurate genomic integration of the donor DNA.
Together, these results highlight the potential of VpCAST as a programmable platform for functional gene knock-in and bacterial genome editing.
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