Twin prime editing expands to gene knockouts and combined precision editing in rice, maize, and wheat
Background
A study published in Nature Biotechnology used twin prime editing to disable genes in rice, maize, and wheat, and combined knockouts with other forms of precision editing. Its central system, twin prime editing-based knockout (TKO), inserts stop-codon clusters (SCCs). The team began by disabling multiple genes at once, then expanded the editing range to precise sequence changes and insertion of large DNA fragments.
A knockout disables a gene so it cannot produce a normal protein. However, an in-frame mutation can leave the protein’s reading frame intact after editing, making the intended loss of function harder to achieve. TKO was designed to insert grouped stop codons that terminate translation and minimize in-frame mutations. The study addressed the difficulty of introducing different types of edits into crop genomes at the same time.
Key findings
In protoplasts—plant cells with their cell walls removed—TKO knockout efficiency reached up to 70.5% in rice, 58.6% in maize, and 75.1% in wheat. In 96.8% of regenerated rice plants, the researchers obtained heritable knockout alleles. This percentage is a result from those regenerated plants; it does not represent the efficiency of every editing type or the transmission rate across all progeny.
In hexaploid wheat, the researchers compared TKO with Cas9 for knocking out all three homologous genes. TKO was 4.2 times as effective at producing this knockout as Cas9, with the researchers attributing the difference mainly to fewer in-frame mutations. This comparison concerns a specific knockout result in hexaploid wheat. The evidence does not support extending it to all editing in rice and maize or to performance differences across prime editing approaches in general.
TKO editors using different SCC sequences enabled simultaneous knockout of up to 10 genes without cross-interference. The available abstract, however, does not specify the experimental material and conditions for this result.
TRIM1 combines TKO with conventional prime editing to perform knockouts and precise edits together. In rice, its joint editing rate across four genes was 22.8%. TRIM2, which combines a prime editor with a recombinase, extended the approach to kilobase-scale editing. In protoplasts, its efficiency for inserting 4.9-kilobase (kb) DNA was 1.2%, while its gene knockout efficiency reached up to 79.8%. The abstract alone does not allow these two figures to be interpreted as rates of achieving both insertion and knockout in the same cells.
Meaning and outlook
This study presents a crop research tool for combining loss of function in multiple genes with other types of precision editing. The recovery of heritable knockout alleles in regenerated rice provides a basis for carrying cell-level editing into plant studies. TRIM2 showed the potential to insert large DNA fragments, but the reported insertion efficiency was 1.2%. Its possible uses need to be assessed in light of both insertion size and efficiency.
The available material is limited to bibliographic information and the abstract. Sample sizes, efficiencies by target site, statistical analyses, and off-target results could not be verified. Improved drought tolerance, nutritional content, or field yields were not among the outcomes reported in the abstract. Further validation is needed before the performance of these editing tools can be linked to agricultural efficacy or the readiness of commercial varieties.
The findings can be checked in the bibliographic record and abstract indexed in Europe PMC.
Published online: June 5, 2026, in Nature Biotechnology. DOI: 10.1038/s41587-026-03174-5
Summary: This pioneering study addresses longstanding inefficiencies and structural insertion limitations in multi-locus genome editing within monocot crops by deploying a Twin Prime Editing infrastructure. By integrating a dual-pegRNA strategy enabling simultaneous template-directed reverse transcription across complementary strands, the computational platform optimizes multi-gene editing kinetics. Experimental validation demonstrated a robust threefold enhancement in precise editing velocities for insertion, deletion, and replacement operations over conventional prime editing methods, successfully integrating traits for drought resilience and nutritional enhancement in standard rice and maize populations. This advancement provides a non-invasive computational framework to minimize false-positive structural variants, calculate multi-gene editing probabilities, and guide universal agricultural stratification strategies.
For studies that compare changes in plants after disabling several candidate genes at once, TKO could serve as a candidate multiplex knockout tool. The reported simultaneous knockout of up to 10 genes supports this potential use. Researchers planning to test loss of function alongside a specific sequence change can also look to TRIM1, which recorded a 22.8% joint editing rate across four genes in rice.
TRIM2 could be considered for experiments that insert large DNA fragments, although its 1.2% efficiency for 4.9-kb insertions was measured in protoplasts. To assess practical use, researchers would need to verify editing outcomes in the target crop and at the intended sites, then evaluate inheritance and traits in regenerated plants. The reported results support gene-function studies and development of combined editing tools; they do not demonstrate improved yields or environmental stress tolerance.