Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis

Trevor Weiss; Maris Kamalu; Honglue Shi; Zheng Li; Jasmine Amerasekera; Zhenhui Zhong; Benjamin A Adler; Michelle M Song; Kamakshi Vohra; Gabriel Wirnowski; Sidharth Chitkara; Charlie Ambrose; Noah Steinmetz; Ananya Sridharan; Diego Sahagun; Jillian F Banfield; Jennifer A Doudna; Steven E Jacobsen

doi:10.1038/s41477-025-01989-9

Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis

Nat Plants. 2025 May;11(5):967-976. doi: 10.1038/s41477-025-01989-9. Epub 2025 Apr 22.

Authors

Trevor Weiss¹, Maris Kamalu¹, Honglue Shi^{2

3}, Zheng Li¹, Jasmine Amerasekera¹, Zhenhui Zhong^{1

4}, Benjamin A Adler^{2

5}, Michelle M Song¹, Kamakshi Vohra^{2

5}, Gabriel Wirnowski¹, Sidharth Chitkara¹, Charlie Ambrose¹, Noah Steinmetz¹, Ananya Sridharan¹, Diego Sahagun¹, Jillian F Banfield^{2

6

7

8}, Jennifer A Doudna^{2

3

5

9

10

11

12

13

14}, Steven E Jacobsen^{15

16}

Affiliations

¹ Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA.
² Innovative Genomics Institute, University of California, Berkeley, CA, USA.
³ Howard Hughes Medical Institute, University of California, Berkeley, CA, USA.
⁴ Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Sciences, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu, China.
⁵ California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA.
⁶ Department of Earth and Planetary Science, University of California, Berkeley, CA, USA.
⁷ Department of Environmental Science, Policy and Management, University of California, Berkeley, CA, USA.
⁸ University of Melbourne, Melbourne, Australia.
⁹ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
¹⁰ Department of Chemistry, University of California, Berkeley, CA, USA.
¹¹ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
¹² Li Ka Shing Center for Translational Genomics, University of California, Berkeley, CA, USA.
¹³ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.
¹⁴ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
¹⁵ Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA. jacobsen@ucla.edu.
¹⁶ Howard Hughes Medical Institute (HHMI), University of California at Los Angeles, Los Angeles, CA, USA. jacobsen@ucla.edu.

Abstract

Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation¹. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Arabidopsis* / genetics
CRISPR-Cas Systems
Gene Editing* / methods
Genome, Plant
Plant Viruses* / genetics
Plants, Genetically Modified / genetics
RNA, Guide, CRISPR-Cas Systems* / genetics
Transgenes

Substances

RNA, Guide, CRISPR-Cas Systems

Supplementary concepts

Tobacco rattle virus

Associated data

BioProject/PRJNA1124592
BioProject/PRJNA1146711

Grants and funding

2334027/National Science Foundation (NSF)