Emerging tools in plant genome editing

Shilpi Sharma^1,2Naveen Kumar Saroha²Abhilasha Sehrawat²Guiliang Tang^3*Deepali Singh^4*Sachin Teotia^2,5*

• ¹Department of Botany, Faculty of Science, University of Delhi, Delhi, NCT of Delhi, India
• ²Department of Biotechnology, Sharda University, Greater Noida, Uttar Pradesh, India
• ³Department of Biological Sciences, Michigan Technological University, Houghton, Michigan, United States
• ⁴School of Biotechnology, Gautam Buddha University, Greater Noida, Uttar Pradesh, India
• ⁵YMCA University of Science and Technology, Faridabad, Haryana, India

Plant genome editing has undergone a transformative shift with the advent of advanced molecular tools, offering unprecedented levels of precision, flexibility and efficiency in modifying genomes. While classical site-directed nucleases such as ZFNs, TALENs and CRISPR-Cas9 have revolutionized genome engineering by enabling targeted mutagenesis and gene knockouts, the landscape is now rapidly evolving with the emergence of novel systems that go beyond the conventional double-strand break (DSB)-mediated approaches. Advanced and recent tools include LEAPER, SATI, RESTORE, RESCUE, ARCUT, SPARDA, helicase-based approaches like HACE and Type IV-A CRISPR system, and transposon-based techniques like TATSI and piggyBac. These tools are unlocking previously inaccessible avenues of genome and transcriptome modulation. Some of these technologies allow DSB-free editing of DNA, precise base substitutions and RNA editing without altering the genomic DNA, a significant advancement for regulatory approval and for species with complex genomes or limited regeneration capacity. While LEAPER, RESCUE and RESTORE are the new advents in the RNA-editing tool, SATI allows DSB-free approach for DNA editing, ARCUT offers less off-target and cleaner DNA repairs and Type IV-A CRISPR system induces gene silencing rather than editing. The transposon-based approaches include TATSI, PiggyBac and TnpB and helicases are used in HACE and Type IV-A CRISPR system. The prokaryotic Argonaute protein is used in SPARDA tool as an endonuclease to edit DNA. The transient and reversible nature of RNA editing tools such as RESTORE and LEAPER introduces a new layer of epigenetics-like control in plant systems which could be harnessed for tissue specific and environment responsive trait expression. Simultaneously, innovations like ARCUT and SPARDA utilize chemically guided precision minimizing reliance on biological nucleases and reducing off target risks. Their modularity and programmability are enabling gene function studies, synthetics pathway designs and targeted trait stacking. These advances represent a novel synthesis of genome engineering and systems biology positioning plant genome editing not just as a tool of modification but as a platform for designing adaptive crops tailored to environmental and nutritional challenges. Although, many of these recent tools remain to be applied on plant systems, they hold a great potential to be effective in creating climate-resilient crops.