CRISPR-Associated Transposase System Can Insert Multiple Copies of Donor DNA into the Same Target Locus

The CRISPR-associated transposase system enables site-specific DNA integration on the genome independent of homologous recombination. Previous studies have demonstrated that the type V-K CRISPR-associated Tn7-like transposase system from Scytonema hofmanni and the type I-F system from Vibrio cholerae have strong target immunity like Tn7, and therefore two or more copies of the donor DNA would not be inserted into the same target location in theory. In this paper, we report that the type I-F system can insert multiple donor copies into one site, which was identified and confirmed by single-strain identification and high-throughput sequencing. This result is beneficial for our application of multicopy chromosomal integration by CRISPR-associated transposases, allowing more donor insertions into the chromosome. This unexpected result shows that the target immunity mechanism of this system has not been fully understood. Attention should be paid to the possibility of multiple insertions and their effects in related research.