Transgene-free Genome Editing in Poplar

Abstract

Achieving precise, transgene-free genome editing in perennial tree species remains a major challenge due to limitations in transformation efficiency, delivery methods, and regulatory concerns. In this study, we developed and evaluated genome editing strategies to optimize transgene-free genome editing in Populus tremula × alba. We compared the performance of two CRISPR-Cas9 base editor vector constructs—one standard (pLR5478) and one tagged with a mobile tRNA-like sequence (TLS; pLR5479)—for editing two key target genes: ALS (acetolactate synthase) and 4CL1 (4-coumarate-CoA ligase 1). Both vectors successfully generated edited plants; however, pLR5478 yielded a higher proportion of transgene-free edited lines compared to pLR5479. Notably, transgene-free plants edited at the 4CL1 locus using the TLS-tagged vector exhibited moderate-to-high editing efficiencies (40.9%–69.97%), outperforming those generated using the standard vector. These findings suggest a trade-off between editing efficiency and transgene-free recovery depending on vector architecture.We further explored biolistic delivery methods using deoxyribonucleic acid (DNA) and ribonucleoprotein (RNP) complexes to the axillary meristem. Comparisons between a low-pressure (650 psi) barrel-based system and a high-pressure (1350 psi) traditional rupture disc method revealed that the barrel system increased transformation efficiency with green fluorescent protein expression. Also, with RNP delivery, more plants were edited using the low-pressure barrel method, but plant samples bombarded using the traditional method had higher indel frequency. These results underscore the importance of optimizing physical delivery parameters to balance cell viability and editing efficacy. In summary, this work demonstrates that TLS-enhanced vectors can improve base editing efficiency at certain loci in poplar but may hinder transgene-free plant recovery. Meanwhile, delivery of RNPs to meristematic tissues, though currently limited in efficiency, offers a viable path toward transgene-free genome editing. Thus, these results provide important insights into the development of precise, DNA-free genome editing systems in tree species and highlight practical trade-offs in vector design and reagent delivery that must be optimized for future deployment in forestry and bioenergy applications.