Twin Prime Editing Enables Rapid Trait Stacking in Crops

Researchers working to advance genome engineering in crops face many challenges, including simultaneously introducing diverse genome edits. Although a major goal of modern crop breeding is to efficiently combine multiple desirable traits by “stacking” the favorable alleles that contribute to those traits in a single crop variety, current strategies are time-consuming and inefficient.

Now, a team led by Caixia Gao, PhD, professor at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences, has developed a genome engineering platform that allows multiple trait stacking in crops by combining gene knockout, precise sequence editing, and chromosome engineering within a single framework. The advance is “a twin prime editing-based knockout (TKO) system that installs stop codon clusters (SCCs) for precise translational termination with minimal in-frame mutations.” TKO achieved knockout efficiencies of up to 70.5%, 58.6% and 75.1% in rice, maize, and wheat protoplasts, respectively.

This work was published in Nature Biotechnology in the article, “Multiplexed, precise genome engineering in monocots with twin prime editing systems.”

The researchers first developed a precise and efficient gene knockout tool called twin prime editing (twinPE)-mediated gene knockout (TKO), which precisely inserts a small fragment containing a stop codon cluster at the target site. TKO achieves predictable gene disruption through precise installation of stop codons, avoiding in-frame indels caused by insertions or deletions in multiples of three nucleotides, which are often seen in Cas9 systems.

In protoplasts, TKO demonstrated efficient knockout capabilities in monocot crops such as rice, wheat, and maize. In regenerated T0 rice plants, the average efficiency for single gene knockout reached 96.8%.

To eliminate cross-editing between different loci and to achieve precise, safe multiplex gene knockout, the researchers developed 10 orthogonal TKO systems, enabling efficient simultaneous knockout of up to 10 genes. Unlike Cas9-mediated multiplex editing, which can lose effectiveness because in-frame mutations accumulate across multiple targets, the orthogonal TKO systems maintain high knockout efficiency even when multiple genes or homologous gene copies are edited simultaneously.

Building on TKO, the researchers then developed two integrated genome engineering platforms, TRIM1 and TRIM2—forming a unified platform known as TRIM.

TRIM1 combines TKO with prime editing-based sequence modification, enabling simultaneous gene knockout, base substitution, insertion, deletion, duplication, and inversion within a single editing framework. In regenerated T0 rice plants, TRIM1 achieved simultaneous knockout of one gene together with homozygous precise editing of three additional targets with an efficiency of 22.8%.

TRIM2 incorporates a prime editor–Cre recombinase fusion protein and enables kilobase-scale DNA insertion, replacement, deletion, inversion, and chromosomal translocation through recombinase-assisted genome engineering.

Unlike existing genome editing tools that typically perform only a limited number of sequence modifications, TRIM integrates gene knockout, small-scale precise sequence editing, and large-scale chromosome engineering into a single platform. This “all-in-one” platform provides a powerful way to rapidly stack multiple favorable alleles, thus enhancing precision breeding of complex traits in monocot crops.

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