Transposase-assisted target-site integration for efficient plant genome engineering

Targeted integration of transgenes in plants remains inefficient and often error-prone, limiting precise genome engineering for crop improvement. Traditional transgenesis inserts DNA randomly, causing unintended mutations and position effects. Attempts at site-specific integration in plants have struggled with low efficiencies and deletions associated with repair pathways such as non-homologous end joining. The study aims to create a high-efficiency, accurate, and programmable system for inserting custom DNA sequences at defined genomic sites by harnessing the natural cut-and-paste capability of class II DNA transposons combined with CRISPR-guided nucleases.

Multiple plant genome engineering strategies have been explored: homologous recombination (HR) is demonstrable but extremely rare in plants and not practical. CRISPR-Cas nucleases increase targeted integration but rely on repair pathways that often introduce indels. Prime editing can insert small sequences (up to ~34 bp) but is insufficient for larger trait-encoding cassettes. Homology-directed repair (HDR) operates at low frequency in plants because double-strand breaks are primarily repaired by non-homologous end joining (NHEJ), which can also cause random DNA integration. NHEJ-based knock-ins can occur but often suffer deletions at flanks and cargo. Prokaryotic CRISPR-associated transposases have been engineered for programmable DNA insertion in bacteria, and synthetic fusions of transposases with programmable nucleases have been tested in animal cells. The natural mPing/Pong transposon system from rice efficiently excises and inserts into genomes and prefers TTA/TAA target-site duplications, suggesting potential for targeted integration when coupled to sequence-specific DSBs.

The authors engineered a transposase-assisted target-site integration (TATSI) system by combining the rice Pong transposase (ORF1 and ORF2) with programmable nucleases (Cas9 or Cas12a). They created 12 fusion protein configurations by fusing Cas9 (active, nickase D10A, or dCas9) to the N- or C-termini of ORF1 and ORF2, alongside unfused controls with ORF1/ORF2 and Cas9 co-expressed. A two-step transformation in Arabidopsis was used: a donor line carried an mPing element (430 bp) embedded within a GFP cassette (to assay excision by restored fluorescence), and a second transgene introduced ORF1/ORF2 with Cas9 and a gRNA targeting PDS3. Excision was confirmed by fluorescence and PCR; targeted insertions were assayed by PCR across junctions using primers in PDS3 and mPing, and validated by Sanger sequencing. Programmability was tested by swapping gRNAs to target ADH1 (exon) and the ACT8 upstream intergenic region, including multiplexing with dual gRNAs. Amplicon deep sequencing of pooled targeted insertion junctions quantified precision and polymorphisms at insertion sites. Genome-wide insertion profiling used insertion-seq: genomic libraries in plasmid vectors followed by PCR between vector and mPing primers and deep sequencing, enabling sensitive detection of rare insertions compared to whole-genome sequencing. A one-component Arabidopsis system was also built with the mPing donor on the same transgene as ORF1/ORF2/Cas9/gRNA to simplify delivery and measure per-plant targeted insertion rates at the ACT8 upstream site. Cargo capacity was tested by embedding: (1) a 6× heat shock element enhancer array (mPing_HSE, 444 bp), (2) the bar coding sequence alone (mPing_bar_CDS, 1,002 bp), (3) a full bar expression cassette with NOS promoter and terminator (mPing_bar, 1,563 bp), and (4) an ~8.6 kb dual-gene cargo (mPing_EPSPS; total 8,994 bp). Targeted insertions and integrity were validated by PCR and Sanger sequencing. For soybean (Glycine max, Williams 82), seven TATSI configurations targeted safe-harbor site DD20 using either ORF2-Cas9 fusions with different G4S linker lengths or unfused ORF2 + Cas9; outcomes measured included mPing excision, Cas9 mutagenesis, and targeted insertion. Junction precision in soybean was evaluated by Sanger sequencing, and insertion-seq in a regenerated plant mapped genome-wide insertion sites. Functional selection was demonstrated by configuring mPing_bar as the sole selectable marker to recover herbicide-resistant transformants after targeted insertion.

• Two configurations enabled robust targeted insertion in Arabidopsis: ORF2-Cas9 fusion and unfused Cas9 co-expressed with ORF1 and ORF2. Targeted insertion required catalytically active Cas9 and both ORF1 and ORF2.
• Programmable nucleases were interchangeable: both ORF2-Cas9 and ORF2-Cas12a (and unfused nucleases) supported mPing excision and targeted insertion, indicating TE insertion into CRISPR-induced double-strand breaks.
• Precision at target sites (PDS3): among >1,703 distinct junctions, most insertions occurred at the CRISPR cut or within 4 bp; 66.8% of insertions had intact mPing ends (full or near-full delivery). Junctions often displayed small NHEJ-associated polymorphisms (1–3 bp indels and occasional deletions up to 7 bp) at the flanking TTA/TAA and target DNA. Unfused configuration yielded slightly more perfect base-for-base insertions than fused (P<0.01).
• Off-target analysis: insertion-seq showed numerous free transpositions in +ORF1/+ORF2 without CRISPR. Adding CRISPR reduced free-transposition sites and enriched the targeted site. Off-target insertions were not at predicted gRNA off-targets and were not shared between replicates, indicating they reflect free transposition rather than CRISPR off-target cleavage. Considering only gRNA-similar sites, TATSI off-target rate is comparable to other state-of-the-art plant target-site integration methods.
• Programmability: Successful targeting to ADH1 exon and upstream of ACT8; multiplexing from a single transcript yielded insertions at both ADH1 and ACT8.
• One-component Arabidopsis system (ACT8 upstream target): ORF2-Cas9 fusion yielded 75.0% excision and 6.7% targeted insertion among 120 T1 plants; unfused ORF2 + Cas9 yielded 98.7% excision and 35.5% targeted insertion. Among plants with excision, 36–45% had targeted insertion, indicating excision is likely rate-limiting. A HITI-like strategy showed intermediate targeted insertion relative to fused and unfused TATSI.
• Cargo delivery in Arabidopsis: mPing_HSE (444 bp) targeted insertion confirmed with all six HSEs intact; mPing_bar_CDS (1,002 bp) and mPing_bar (1,563 bp) targeted insertion in 26.7% and 27.7% of T1 plants, respectively, with intact cargo sequences; very large cargo (mPing_EPSPS, 8,994 bp) targeted insertion at 8.3%, indicating efficiency decreases with larger cargos.
• Soybean DD20 safe harbor: ORF2-Cas9 fusion with 1×G4S linker lacked Cas9 activity; 3×G4S linker restored activity (excision 76.2%, Cas9 mutations 38.1%, targeted insertion 15.9%). Unfused ORF2 + Cas9 achieved excision 92.7% and targeted insertion 18.2%. Junctions showed small NHEJ deletions typical of TATSI. Insertion-seq in a regenerated plant revealed seven major mPing insertions including the targeted DD20 site; other insertions occurred at TTA/TAA motifs (free transposition). mPing_HSE and mPing_bar cargos also targeted in soybean with 6.3% and 6.7% targeted insertion, respectively. Configuring mPing_bar as the sole selectable marker yielded herbicide-resistant plants with 94.1% excision and 9.8% targeted insertion; in some cases, the targeted insertion was present without the parent donor transgene.

By fusing or co-expressing rice Pong transposase proteins with CRISPR nucleases, the TATSI system directs the natural cut-and-paste ability of mPing into CRISPR-generated double-strand breaks, achieving programmable, sequence-specific insertion of DNA cargos in planta. The approach addresses the low efficiency and error-prone nature of HDR- and NHEJ-based targeted insertions by protecting cargo ends during extrachromosomal phases and channeling insertion to a defined break. The system improves on-target frequencies over established HR/HDR/NHEJ methods in Arabidopsis, and matches or exceeds state-of-the-art targeted insertion rates reported for soybean, while maintaining low apparent CRISPR off-target insertion rates. The high integrity of delivered cargos and predominance of small, local NHEJ junction edits make TATSI suitable for precise trait introduction, enhancer delivery, and potential trait stacking. The presence of Cas9 reduces free transpositions, and targeted insertions often disrupt the flanking TTA/TAA duplication needed for excision, favoring stability of the inserted cargo. The technology is likely broadly applicable across transformable plants, with selection of appropriate TE systems to avoid identity-based silencing in the host species.

The study introduces TATSI, a practical plant genome engineering toolkit that combines a DNA transposase with CRISPR nucleases to achieve efficient, accurate, and programmable insertion of custom DNA sequences in Arabidopsis and soybean. It demonstrates high on-target rates, broad cargo capacity (from enhancers to multi-kilobase gene cassettes), multiplex targeting, and compatibility with one-component delivery. TATSI outperforms or rivals existing targeted integration strategies and is well-suited for crop improvement applications. Future work should focus on increasing excision rates (for example, using hyperactive mPing variants), controlling insertion orientation, further minimizing free transpositions, optimizing fusion linkers and protein configurations across species, and expanding to additional crop genomes with appropriately chosen TE systems.

• Insertion orientation is currently uncontrolled.
• Free transpositions occur due to active mPing, though reduced by the presence of CRISPR-induced DSBs.
• Small NHEJ-associated indels at insertion junctions are common.
• Fusion of ORF2 to Cas9 reduces overall activity (excision and insertion rates), though it increases the proportion of on-target insertions.
• Alterations to mPing sequence and larger cargos reduce targeted insertion efficiency.
• The rice mPing/Pong system may be epigenetically silenced in rice; species-specific TE systems may be needed.