Development of a highly efficient prime editing platform for cucurbits enables breeding of multi-disease-resistant cucumber.

Establishment of an efficient and PAM-relaxed LbCas12a genome editing tool in plants.

Cas12a is widely used in plant genome editing, but its targeting scope is constrained by stringent protospacer adjacent motif (PAM) requirements and variable activity across species, limiting its application at diverse genomic loci. LbCas12a-RRV-based editing system was established in nonheading Chinese cabbage, and T5exo-PF-LbCas12a was generated by introducing a triple mutation (D535G/S551F/D665N) and fusing with T5 exonuclease. This engineered system recognizes an expanded PAM sequence from 5'-VTTV-3' to 5'-NYHV-3'. The system exhibited efficient editing at noncanonical PAM sites in cabbage, tomato, and rice. Additionally, it successfully mediated large-fragment deletions via microhomology-mediated end joining (MMEJ) in plants. This study expands Cas12a targeting scope in plants and provides the first evidence for Cas12-mediated MMEJ-based large-fragment deletion. The toolkit facilitates functional genomics and crop improvement, and the methodology is readily adaptable to other plant species.

A Robust Framework for Maize Elite Line Genome Editing Through Enhanced HI‐Edit via LbCas12a Activity Optimization

ABSTRACT Haploid induction coupled with genome editing (HI‐Edit) enables direct modification of commercial crop varieties, bypassing the need for trait introgression or direct transformation of elite lines with CRISPR machinery. However, its widespread application has been constrained by low haploid editing rates (HER), the proportion of haploids carrying edits within the short window between double fertilization and uniparental chromosome elimination. Here, we report substantial improvements in maize HI‐Edit efficiency through three complementary strategies: (1) driving an optimized LbCas12a variant (LbCas12aV) using promoters that are highly active in sperm cells and early zygotes; (2) applying a post‐pollination heat treatment; and (3) fusing LbCas12aV with the UBA2 domain (ubiquitin‐associated domain‐2 of Arabidopsis thaliana RAD23) to enhance protein stability during haploid induction. Post‐pollination heat treatment alone increased HER to 19.1% (up to 12‐fold improvement depending on the target site), providing a simple and effective method to boost the yield of edited doubled haploid (DH) plants. UBA2 fusion improved HER by 6‐fold at the Waxy1 (Wx1) locus and 4.5‐fold at the Glossy2 (Gl2) locus under normal conditions. Strikingly, combining UBA2 fusion with heat treatment raised the average HER to 25% across multiple events targeting Wx1, with the highest HER reaching 33%. Collectively, these findings demonstrate that increasing CRISPR‐Cas protein abundance and modulating environmental conditions can overcome key bottlenecks in HI‐Edit. We establish a robust, scalable framework that is readily transferable to other crops for elite‐line genome editing.

CRISPR/Cas9-Mediated Editing of Bsr-d1 and Pi21 Enhances Blast Resistance in a High-Quality Rice Maintainer Line

Rice (Oryza sativa L.) is a staple food crop worldwide, and improving disease resistance is a core target in rice breeding. In this study, we employed CRISPR/Cas9 genome editing to modify the coding sequence (CDS) of two susceptibility genes, Bsr-d1 and Pi21, in the elite maintainer line Gengxiang B to enhance its blast resistance. We generated Bsr-d1/Pi21 double homozygous mutants via Agrobacterium-mediated genetic transformation. Quantitative RT-PCR revealed significantly suppressed transcript accumulation of both target genes in the edited lines compared with the wild type Gengxiang B. Upon inoculation with Magnaporthe oryzae, multiple defense-related marker genes were markedly upregulated in the double mutants. Phenotypic assays demonstrated significantly reduced disease severity for both leaf and panicle blast in the edited lines compared with the wild type. Importantly, no statistically detectable differences were found between the double mutants and wild-type plants for key agronomic or grain quality traits. Collectively, these results demonstrate that CRISPR/Cas9-mediated editing of susceptibility loci generates genetically stable blast-resistant rice germplasm without compromising agronomic traits or grain quality, providing valuable genetic resources for future rice varietal improvement.

A Novel Genome Editing Strategy in Plants Using Broad-Host-Range Viral Vectors Derived from Geminiviruses.

The use of viral vectors offers a promising alternative to traditional transformation methods for creating gene-edited plants. In this study, we developed a novel plant genome editing system by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB). Cas9, Cas12f, and Cas12j nucleases were efficiently expressed along with corresponding guide RNAs under viral promoters. By leveraging tRNA spacers in place of external promoters and terminators, we significantly reduced the overall cargo size, streamlining vector design. Additionally, we compared the traditional AtU6-driven gRNA delivery with a novel spacer:gRNA:spacer format in Cas9-expressing lines and observed comparable editing efficiencies. The broad host range of WDIV and AYLCB, combined with the novel genome-editing platform, opens possibilities for editing across a wide range of plant species.

Development of efficient methods for protoplast-to-plant regeneration in Coffea arabica and successful CRISPR-Cas9 editing in protoplasts

Conventional coffee breeding is a long and complex process that limits genetic improvement, highlighting the need for advanced biotechnological tools. Protoplast-based CRISPR genome editing is a promising transgene-free approach, which enables precise genetic modifications and whole-plant regeneration while avoiding the stable integration of foreign DNA. However, reliable plant regeneration from protoplasts remains a significant challenge in most crops, especially in tropical tree species like coffee. Here, we present an efficient protocol for protoplast isolation from embryogenic calli and protoplast-to-plant regeneration in the allotetraploid species Coffea arabica. Culture conditions were optimised by comparing protoplast-derived cell growth in liquid medium versus alginate layers and by assessing medium supplementation with the peptide growth factor phytosulfokine-α (PSK) or polyamines (spermine and putrescine). The alginate-embedding system, combined with 0.1 µM or 1 µM of PSK, significantly enhanced early cell division frequencies (up to 50%) and facilitated the formation of up to 270 microcalli.cm−2, enabling subsequent whole-plant regeneration using somatic embryogenesis. Although spermine also favoured cell division, its effect was weaker than that of PSK, and no synergistic interaction was observed when combined with PSK. Furthermore, we demonstrated successful polyethylene glycol-mediated transfection of coffee protoplasts with a CRISPR–Cas9 plasmid targeting the coffee xanthosine methyltransferase (XMT) gene involved in caffeine biosynthesis. Subgenome-specific PCR and deep sequencing revealed insertion/deletion mutations at target sites in ca. 4% of protoplasts. Collectively, these findings pave the way for transgene-free genome editing and functional genomics studies based on cutting-edge technologies in coffee.

Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola ‘Sunset Velvet’