Type I-F CRISPR-associated transposons contribute to genomic plasticity in Shewanella and mediate efficient programmable DNA integration

The genome plasticity of species and strains in the genus Shewanella is closely associated with the diverse mobile genetic elements embedded in its genomes. One mobile element with potential for accurate and efficient DNA insertion in Shewanella is the type I-F3 CRISPR-associated transposon (I-F3 CAST). However, relatively little is known about the distribution and ecological significance of I-F3 CASTs and whether they could be suitable as a tool for targeted genetic manipulation in situ. To better understand the distribution of I-F3 CASTs in Shewanella, we analysed 602 Shewanella genomes. We found that I-F3 CASTs were present in 12% of all genomes, although differences in both gene arrangement and integration locus were observed. These Shewanella I-F3 CASTs carried up to 89 cargo genes, which were associated with diverse functions, including defence, resistance and electron transfer, demonstrating an important role in genomic diversification and ecological adaptation. We tested whether the I-F3 CAST present in Shewanella sp. ANA-3 enhanced gene insertion, both in situ and in a heterologous host. We observed I-F3 CAST-mediated crRNA-targeted integration of the supplied genes into the pyrF locus in Shewanella sp. ANA-3. Heterologous gene insertion with high integration efficiency in Escherichia coli was also demonstrated using a simplified version of ANA-3 I-F3 CAST. Altogether, this work highlights the important role of I-F3 CASTs in promoting genomic plasticity of the Shewanella genus and demonstrates the gene-editing capability of ANA-3-CAST both endogenously and heterologously.