Castillo, K. D.; Seephetdee, C.; Horikawa, W.; Kiss, D. L.

Circular RNAs (circRNAs) have emerged as promising platforms for stable RNA expression and therapeutic applications, but methods for their generation remain limited. Here, we engineered the Lactococcus lactis LtrB group II intron into a permuted intron-exon (PIE) configuration to enable efficient circRNA formation (circLtrB). Among the tested split designs, LtrB domain IV cleavage yielded the highest circularization efficiency. Although LtrB encodes LtrA maturase required for efficient in vivo splicing, in vitro application of LtrA to the PIE constructs resulted in lower circularization efficiency than self-splicing conditions. We further examined circularization under distinct salt conditions and showed that NH4Cl- and KCl-based splicing buffers independently support high circularization efficiencies (>80%), while producing distinct splicing intermediates, indicating ion-specific mechanistic effects on circularization. Extending outer homology arms did not enhance circularization efficiency, demonstrating that efficient circRNA formation in the circLtrB platform is independent of extended exon pairing interactions. Using this system, we generated circRNAs across a range of coding sequence lengths, highlighting its payload flexibility. To optimize translation, we evaluated multiple internal ribosome entry sites (IRES), including Aichivirus, Crohivirus, CVB3, improved EMCV, HRV, and Salivirus, and found that the CVB3 IRES drove the highest translation efficiency. Linear NLuc showed faster decay kinetics, while circLtrB-NLuc exhibited more sustained expression over time. Transcriptome-wide RNA sequencing revealed comparable innate immune responses between circular and linear RNAs. Together, these results establish circLtrB as a versatile and efficient platform for circRNA production, providing mechanistic insight into group II intron-mediated circularization and expanding the toolkit for functional circRNA engineering.