Institute of Genetics and Developmental Biology, CAS Develops TKO and "All-in-One" TRIM Genome Editing System, Achieving "One-Stop" Precise Pyramiding of Complex Crop Traits

In the process of modern agricultural crop breeding, although a large number of elite alleles have been successively sequenced and identified, how to rapidly and precisely pyramid them into a single variety has always faced the technical bottleneck of multi-generational crossbreeding and repeated experimental optimization. The research team at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (CAS) has successfully tackled this challenge by developing a precise gene knockout tool TKO and an “all-in-one” composite genome editing system TRIM (Targeted Replacement, Insertion and Mutagenesis), opening up a new pathway for “one-stop” targeted improvement of complex agronomic traits.

To address the challenges of lengthy cycles and limited efficiency in conventional gene editing for polyploid crops and multi-gene coordinated improvement, the research team developed TKO, a highly efficient knockout tool that targets site-specific insertion of stop codon clusters, based on a twin prime editing strategy (twinPE). This tool can achieve deterministic inactivation of target genes by precisely writing stop codons, thereby mechanistically circumventing the issue of in-frame mutations that are easily introduced by traditional CRISPR/Cas9 nucleases due to non-homologous end joining (NHEJ). Experiments showed that TKO exhibited excellent editing activity in protoplasts of major monocot crops including rice, wheat, and maize, with an average single-gene knockout efficiency of up to 96.8% in regenerated T0 rice plants. Ten mutually orthogonal TKO systems constructed on this basis successfully achieved simultaneous precise knockout of up to 10 genes in a single plant.

To further overcome the limitation that different types of gene editing rely on separate tools, the team proposed an “all-in-one” editing architecture and successively developed two integrated systems, TRIM1 and TRIM2. In validation using T0 rice plants, TRIM1 simultaneously achieved knockout of 1 gene and homozygous precise editing of 3 different genes, with a composite editing efficiency of 22.8%; the further upgraded TRIM2 system not only retained all the knockout and base editing capabilities of TRIM1, but also enabled higher-order genome restructuring manipulations including kilobase (Kb)-level large fragment DNA site-specific insertion, replacement, deletion, inversion, and chromosomal translocation.

To substantially lower the barriers to bioinformatics design and wet-lab vector construction, the research team simultaneously developed the supporting automated design software TKO_PegDesigner and established a two-round ligation-based high-efficiency multi-(pe)gRNA assembly strategy, enabling high-throughput assembly of up to 36 (pe)gRNAs in a single reaction. The TRIM system, for the first time internationally, integrates gene inactivation knockout, precise nucleotide editing, and large-scale chromosomal engineering restructuring onto the same technological platform, providing powerful foundational engineering tool support for targeted molecular design breeding of major grain and oil crops and large-scale pyramiding of key elite traits.