Plant cell reportsFeb 17, 2026

Genetic editing without added genes in citrus and poplar growing tissues using direct CRISPR-Cas9 protein delivery

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 27, 2026

Abstract

Essence

Biolistic CRISPR-Cas9 RNP delivery can create transgene-free edits in woody plant meristems.

Evidence

In a plant genome-editing platform experiment, RNP bombardment of citrus shoot apical and poplar axillary meristems generated CsNPR3 and Pt4CL1 edits, while citrus plasmid vectors failed.

Caveat

Detected chimeric events mean the approach is not yet shown to reliably produce fully edited, uniform perennial plants.

Simplified

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.

Full Text

What this is

This research explores a novel method for genome editing in citrus and poplar plants using CRISPR-Cas9.
Biolistic particle bombardment delivers ribonucleoprotein complexes into meristem tissues, which are crucial for plant regeneration.
The approach successfully generates targeted mutations without introducing foreign DNA, addressing challenges in conventional tissue culture.

Essence

Biolistic delivery of CRISPR-Cas9 ribonucleoprotein complexes enables targeted genome editing in citrus and poplar meristem tissues, producing transgene-free edited plants.

Key takeaways

In poplar, successful genome editing occurred at the Pt4CL1 gene using the same RNP approach.
This technique provides a feasible framework for producing edited perennial plants without transgenes.

Simplified

Supplementary Information

Below is the link to the electronic supplementary material.

Acknowledgements

We thank Colby Starker and Daniel Voytas for sharing the pCGS710 vector. We thank members of the Qi lab for their feedback in the development of this project. This research was partly supported by the NSF Plant Genome Research Program (award no. IOS-2132693 and IOS-2428015), USDA-NIFA Agricultural Innovation through Gene Editing (award no. 2021-67013-34554), USDA-NIFA Biotechnology Risk Assessment Research (2024-33522-42755), USDA-NIFA’s Emergency Citrus Disease Research and Extension (award no. 2020-70029-33161), Department of Energy (award no. award no. DE-SC0023011) and McIntire Stennis Forest Research Program (MD-PSLA-24014). The authors acknowledge FAPESP for granting a scholarship to author Dhiôvanna Corrêia Rocha (process numbers: 2020/07045-3, 2021/03466-7, and 2023/09068-9).

Author contributions

YQ proposed and supervised the project. DCR, MOO, and WL designed the experiments. DCR generated the CRISPR-Cas9 vectors. DCR and MOO performed the genetic transformation of citrus and poplar, respectively. DCR, MOO, and JM worked on plant regeneration. HF generated the Cas9 expression vector for E.coli expression. QY and YL purified Cas9 protein from E.coli. WL taught DCR and MOO the SAM exposed procedure and supported the biolistic experiments. JC provided technical support to citrus transformation. GL and GC provided material and technical support to poplar transformation. AAS provided resources to the project. DCR MOO, and YQ analyzed the data and wrote the manuscript with input from other authors. All authors read, revised, and approved the final manuscript.

Funding

Fundação de Amparo à Pesquisa do Estado de São Paulo, 2020/07045-3,2021/03466-7, and 2023/09068-9, U.S. Department of Energy, DE-SC0023011, Directorate for Biological Sciences, IOS-2132693 and IOS-2428015, National Institute of Food and Agriculture, 2020-70029-33161, 2021-67013-34554, 2024-33522-42755, McIntire Stennis Forest Research Program, MD-PSLA-24014

Data availability

All our data were included in the main figure and supplementary information. Request for the raw data should be communicated to the corresponding author.

Declarations

Conflict of interest

The authors declare no competing interests.

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

All our data were included in the main figure and supplementary information. Request for the raw data should be communicated to the corresponding author.

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Genetic editing without added genes in citrus and poplar growing tissues using direct CRISPR-Cas9 protein delivery

Abstract

Full Text

What this is

Essence

Key takeaways

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Conflict of interest

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

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