# Transgene-free genome editing in citrus and poplar meristem tissues via biolistic ribonucleoprotein delivery of CRISPR-Cas9

## TL;DR

Researchers used a new method to edit the genomes of citrus and poplar plants without leaving transgenes, using CRISPR-Cas9 delivered directly into plant cells.

## Contribution

A novel transgene-free CRISPR-Cas9 delivery method using biolistic RNP delivery in meristem tissues of woody plants is introduced.

## Key findings

- CRISPR-Cas9 ribonucleoproteins successfully edited citrus and poplar genomes in meristem tissues.
- Plasmid-based vectors failed in citrus, but RNP delivery succeeded.
- Chimeric events occurred in poplar, but the method remains viable for transgene-free editing.

## Abstract

Biolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes.

The use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants.

The online version contains supplementary material available at 10.1007/s00299-026-03741-9.