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<p>Efficient delivery of CRISPR reagents into plant cells remains a major bottleneck, particularly for transgene-free genome editing. RNA viral vectors such as Tabacco Rattle Virus (TRV) provide an attractive platform for transient expression but are limited by their small cargo capacity. Recently discovered miniature nucleases like Cas12f, due to their compact size and high activity, offer a promising alternative for viral delivery systems. In this study, a plant codon-optimized <i>Acidibacillus sulfuoxidans</i> Cas12f (<i>As</i>Cas12f), fused with nuclear localization signals, was cloned into the TRV genome to develop a viral vector-based delivery platform. Two sgRNAs targeting the <i>Nicotiana benthamiana PHYTOENE DESATURASE</i> (<i>NbPDS</i>) gene were delivered through <i>Agrobacterium</i>-mediated infiltration. To enhance mobility and transcript abundance, mobile RNA elements such as modified <i>Flowering Locus T</i> (mFT), truncated <i>FLOWERING LOCUS T</i> (tFT), and transfer RNA of methionine (tRNA<sup>Met</sup>), and the <i>Pea early browning virus</i> (PeBV) promoter were incorporated into Cas12f and sgRNA constructs. TRV-mediated delivery of CRISPR-Cas12f induced targeted mutagenesis in <i>N. benthamiana</i> systemic leaves, confirmed by persistent photobleaching and Sanger sequencing. Both t<i>FT</i>- and tRNA-tagged Cas12f constructs improved editing efficiency, with the tRNA fusion showing enhanced activity. Expression of Cas12f and sgRNA under the PeBV promoter further enhanced mutation frequency, with pTRV2-PeBV::Cas12f-tRNA and pTRV2-PeBV::mFT-sgRNA constructs achieving the highest efficiency. This study establishes a compact TRV-based CRISPR-Cas12f platform enabling efficient, transgene-free genome editing in plants. The system bypasses tissue culture-dependent transformation and provides a biosafety-compliant strategy for scalable, non-transgenic crop improvement.</p>

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Transgene-free plant genome editing via viral delivery of miniature CRISPR-Cas12f

  • 期刊论文

  • 发表日期：

    2026年6月1日

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  • Muhammad Jawad Akbar Awan,
  • Afzal Akram,
  • Rubab Zahra Naqvi,
  • Mariam Akhtar,
  • Saqib Siddique,
  • Muhammad Ismail Buzdar,
  • Imran Amin,
  • Shahid Mansoor

摘要

Efficient delivery of CRISPR reagents into plant cells remains a major bottleneck, particularly for transgene-free genome editing. RNA viral vectors such as Tabacco Rattle Virus (TRV) provide an attractive platform for transient expression but are limited by their small cargo capacity. Recently discovered miniature nucleases like Cas12f, due to their compact size and high activity, offer a promising alternative for viral delivery systems. In this study, a plant codon-optimized Acidibacillus sulfuoxidans Cas12f (AsCas12f), fused with nuclear localization signals, was cloned into the TRV genome to develop a viral vector-based delivery platform. Two sgRNAs targeting the Nicotiana benthamiana PHYTOENE DESATURASE (NbPDS) gene were delivered through Agrobacterium-mediated infiltration. To enhance mobility and transcript abundance, mobile RNA elements such as modified Flowering Locus T (mFT), truncated FLOWERING LOCUS T (tFT), and transfer RNA of methionine (tRNAMet), and the Pea early browning virus (PeBV) promoter were incorporated into Cas12f and sgRNA constructs. TRV-mediated delivery of CRISPR-Cas12f induced targeted mutagenesis in N. benthamiana systemic leaves, confirmed by persistent photobleaching and Sanger sequencing. Both tFT- and tRNA-tagged Cas12f constructs improved editing efficiency, with the tRNA fusion showing enhanced activity. Expression of Cas12f and sgRNA under the PeBV promoter further enhanced mutation frequency, with pTRV2-PeBV::Cas12f-tRNA and pTRV2-PeBV::mFT-sgRNA constructs achieving the highest efficiency. This study establishes a compact TRV-based CRISPR-Cas12f platform enabling efficient, transgene-free genome editing in plants. The system bypasses tissue culture-dependent transformation and provides a biosafety-compliant strategy for scalable, non-transgenic crop improvement.