Transposition of transposable element IS1 in Edwardsiella piscicida mutant generated by CRISPR/Cas9 along with λ-Red recombineering system.

This study aimed to investigate unintended mutations introduced by the CRISPR/Cas9 genome editing system in Edwardsiella piscicida. Whole-genome sequencing was conducted on the wild-type E. piscicida NH1 and its alanine racemase knockout mutants (E. piscicida Δalr325 NH1 and E. piscicida Δalr50 NH1) generated using CRISPR/Cas9 with a λ-Red recombineering system. Comparative genomic analyses revealed that the insertion sequence 1 (IS1) transpositions occurred in the CRISPR/Cas9-edited mutants, disrupting the type I restriction-modification system subunit M gene, in addition to the targeted gene deletion. Interestingly, no IS1 transpositions were detected in mutants produced via conventional plasmid-based allelic exchange, indicating the potential link between CRISPR/Cas9-mediated editing and transposition events. These results suggest that genome editing via CRISPR/Cas9 could trigger IS1 transposition, potentially due to double-stranded DNA breaks. The lack of sequence similarity between the single guide RNA (sgRNA) and the transposed regions suggests that transpositions are not CRISPR/Cas9 off-target effects. This study provides evidence of interactions between mobile genetic elements and genome editing systems, requiring further investigation into their underlying mechanisms.