Enhanced CRISPR-Cas3-mediated genome editing using circularized crRNAs

Type I-E CRISPR-Cas3 represents a genome-editing technology in which large deletions averaging several kilobases are introduced in target regions. However, its genome-editing efficiency varies considerably across targets and cell types, making it difficult to achieve consistent results. Here, we investigated the efficacy and stability of circularized CRISPR RNAs (ccrRNAs) to enhance CRISPR-Cas3-mediated genome editing in human cells. Using in vitro single-strand DNA cleavage assays, we demonstrated that ccrRNA induces Cascade complex formation. Significant genome-editing activity targeting the EMX1 and B2M genes was observed in cellular assays using K562 cells. Long-read sequencing identified large-scale deletion mutations at the target loci and no detectable off-target effects using ccrRNA. Furthermore, ccrRNAs exhibited extended intracellular stability compared with that for linear crRNAs, resulting in an enhanced editing efficiency. These results demonstrate that ccrRNAs enable stable, efficient, and highly specific genome editing and support the broader application of the long-range deletion system.